Please help
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Hello everybody,
This is my first message to confocal community. I am also molecular biologist:-(( and do not know all tricks in IF. I am trying to understand where is my FLAG tagged protein localized in breast epithelial cells which also has endogenous one. I grow cells in chamber slides. Old lab protocol show strong perinuclear staining for both ABs to our protein and FLAG. Protocol include: 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton for 1 hour. I was suspicious of too long paraformaldehyde treatment and too low Triton percentage. I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and get opposite result. In my variation our protein mostly cytoplasmic and nuclear. We also know that under some condition this protein create filamentous structure. If somebody could help me to understand which protocol is right, what kind of variation of protocol I could use to solve the problem, I would very much appreciate. Thank you, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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Olga,
The triton concentration of 0.025% seems too low and may not work following 40 min fixation in 4% PFA. Try 10 -15 minutes fixation with freshly prepared 2% PFA and 5 min permeabilization with of 0.2% triton. Hope this helps On Wed, May 19, 2010 at 12:26 PM, Olga Makarova <[hidden email]> wrote: > Hello everybody, > > This is my first message to confocal community. I am also molecular biologist:-(( and do not know all tricks in IF. > I am trying to understand where is my FLAG tagged protein localized in breast epithelial cells which also has endogenous one. > I grow cells in chamber slides. > Old lab protocol show strong perinuclear staining for both ABs to our protein and FLAG. Protocol include: > 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. > Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton for 1 hour. > > I was suspicious of too long paraformaldehyde treatment and too low Triton percentage. > > I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and get opposite result. > > In my variation our protein mostly cytoplasmic and nuclear. > > We also know that under some condition this protein create filamentous structure. > > If somebody could help me to understand which protocol is right, what kind of variation of protocol I could use to solve the problem, I would very much appreciate. > > Thank you, > > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > |
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In reply to this post by Olga Makarova
Olga,
In and of itself, paraformaldehyde does not typically require Triton as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % Triton is still a fairly low concentration but maybe enough to punch holes in your cell membranes causing a complete redistribution of intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing which protein you are tagging with FLAG, I can only speculate what the consequences might be. It is a bit unclear what you mean by "opposite results." I am assuming that in the first protocol (low Triton) you get what appears to be colocalization of your protein and the FLAG tag, which is what you logically would hope for. In the high Triton protocol, you lose the peri-nuclear localization, but is this true also for the protein-FLAG version, i.e., do the distributions remain coincident or is that lost? If colocalization remains then one would be tempted to blame the high Triton for possibly mobilizing the target proteins away from the nucleus and into the cytoplasm. If target protein and the FLAG version no longer coincide then things are more complicated. Anyway, using PF without detergent should work fine for fixation and I would tend to believe it before accepting simultaneous PF and Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? For sturdier fixation you might also try 3-4% PF plus 0.2% glutaraldehyde in the initial fix. The PF goes in first and does the initial fixation and allows glut. to follow and reinforce your target crosslinking. With regards to using milk and BSA for blocking, I would seriously consider changing over to an appropriate animal serum appropriate to you antibodies, or better yet a product like Pierce's SuperBlock. Let us know how things go, Mario >Hello everybody, > >This is my first message to confocal community. I am also molecular >biologist:-(( and do not know all tricks in IF. >I am trying to understand where is my FLAG tagged protein localized >in breast epithelial cells which also has endogenous one. >I grow cells in chamber slides. >Old lab protocol show strong perinuclear staining for both ABs to >our protein and FLAG. Protocol include: >40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >for 1 hour. > >I was suspicious of too long paraformaldehyde treatment and too low >Triton percentage. > >I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >get opposite result. > >In my variation our protein mostly cytoplasmic and nuclear. > >We also know that under some condition this protein create >filamentous structure. > >If somebody could help me to understand which protocol is right, >what kind of variation of protocol I could use to solve the problem, >I would very much appreciate. > >Thank you, > >Olga > >Olga Makarova, PhD >Research Specialist >5323 MSRB III > > >********************************************************** >Electronic Mail is not secure, may not be read every day, and should >not be used for urgent or sensitive issues -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] |
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Mario, thank you very much for your response.
I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. My protein is a SEPT9 which is implicated in many cancerous transformations. In both protocols FLAG and protein staining colocolize very well.. Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. Or, vice versa. My protocol does something strange that relocates my protein to nucleus. Thank you, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> Olga, In and of itself, paraformaldehyde does not typically require Triton as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % Triton is still a fairly low concentration but maybe enough to punch holes in your cell membranes causing a complete redistribution of intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing which protein you are tagging with FLAG, I can only speculate what the consequences might be. It is a bit unclear what you mean by "opposite results." I am assuming that in the first protocol (low Triton) you get what appears to be colocalization of your protein and the FLAG tag, which is what you logically would hope for. In the high Triton protocol, you lose the peri-nuclear localization, but is this true also for the protein-FLAG version, i.e., do the distributions remain coincident or is that lost? If colocalization remains then one would be tempted to blame the high Triton for possibly mobilizing the target proteins away from the nucleus and into the cytoplasm. If target protein and the FLAG version no longer coincide then things are more complicated. Anyway, using PF without detergent should work fine for fixation and I would tend to believe it before accepting simultaneous PF and Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? For sturdier fixation you might also try 3-4% PF plus 0.2% glutaraldehyde in the initial fix. The PF goes in first and does the initial fixation and allows glut. to follow and reinforce your target crosslinking. With regards to using milk and BSA for blocking, I would seriously consider changing over to an appropriate animal serum appropriate to you antibodies, or better yet a product like Pierce's SuperBlock. Let us know how things go, Mario >Hello everybody, > >This is my first message to confocal community. I am also molecular >biologist:-(( and do not know all tricks in IF. >I am trying to understand where is my FLAG tagged protein localized >in breast epithelial cells which also has endogenous one. >I grow cells in chamber slides. >Old lab protocol show strong perinuclear staining for both ABs to >our protein and FLAG. Protocol include: >40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >for 1 hour. > >I was suspicious of too long paraformaldehyde treatment and too low >Triton percentage. > >I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >get opposite result. > >In my variation our protein mostly cytoplasmic and nuclear. > >We also know that under some condition this protein create >filamentous structure. > >If somebody could help me to understand which protocol is right, >what kind of variation of protocol I could use to solve the problem, >I would very much appreciate. > >Thank you, > >Olga > >Olga Makarova, PhD >Research Specialist >5323 MSRB III > > >********************************************************** >Electronic Mail is not secure, may not be read every day, and should >not be used for urgent or sensitive issues -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. [hidden email] [hidden email] ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton.
Answer is yes. It is difficult or even impossible to detect nuclear antigens using such an intense fixation with PFA (4%, 40 min) followed by permeabilization with 0.025% triton. As a control you may also try 20 min fixation in methanol on ice (no triton is needed). Methanol fixation optionally may be followed by the immersion of cells in acetone for 10 sec and air dry. Methanol does not preserve the structure as good as PFA does, but it will reveal the distribution of your protein more efficiently. Art On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: > Mario, thank you very much for your response. > > I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. > My protein is a SEPT9 which is implicated in many cancerous transformations. > In both protocols FLAG and protein staining colocolize very well.. > Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. > I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. > Or, vice versa. My protocol does something strange that relocates my protein to nucleus. > > Thank you, > > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> > Olga, > > In and of itself, paraformaldehyde does not typically require Triton > as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % > Triton is still a fairly low concentration but maybe enough to punch > holes in your cell membranes causing a complete redistribution of > intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing > which protein you are tagging with FLAG, I can only speculate what > the consequences might be. > > It is a bit unclear what you mean by "opposite results." I am > assuming that in the first protocol (low Triton) you get what appears > to be colocalization of your protein and the FLAG tag, which is what > you logically would hope for. In the high Triton protocol, you lose > the peri-nuclear localization, but is this true also for the > protein-FLAG version, i.e., do the distributions remain coincident or > is that lost? If colocalization remains then one would be tempted to > blame the high Triton for possibly mobilizing the target proteins > away from the nucleus and into the cytoplasm. > > If target protein and the FLAG version no longer coincide then things > are more complicated. > > Anyway, using PF without detergent should work fine for fixation and > I would tend to believe it before accepting simultaneous PF and > Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? > > For sturdier fixation you might also try 3-4% PF plus 0.2% > glutaraldehyde in the initial fix. The PF goes in first and does the > initial fixation and allows glut. to follow and reinforce your target > crosslinking. > > With regards to using milk and BSA for blocking, I would seriously > consider changing over to an appropriate animal serum appropriate to > you antibodies, or better yet a product like Pierce's SuperBlock. > > Let us know how things go, > Mario > >>Hello everybody, >> >>This is my first message to confocal community. I am also molecular >>biologist:-(( and do not know all tricks in IF. >>I am trying to understand where is my FLAG tagged protein localized >>in breast epithelial cells which also has endogenous one. >>I grow cells in chamber slides. >>Old lab protocol show strong perinuclear staining for both ABs to >>our protein and FLAG. Protocol include: >>40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>for 1 hour. >> >>I was suspicious of too long paraformaldehyde treatment and too low >>Triton percentage. >> >>I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>get opposite result. >> >>In my variation our protein mostly cytoplasmic and nuclear. >> >>We also know that under some condition this protein create >>filamentous structure. >> >>If somebody could help me to understand which protocol is right, >>what kind of variation of protocol I could use to solve the problem, >>I would very much appreciate. >> >>Thank you, >> >>Olga >> >>Olga Makarova, PhD >>Research Specialist >>5323 MSRB III >> >> >>********************************************************** >>Electronic Mail is not secure, may not be read every day, and should >>not be used for urgent or sensitive issues > > > -- > ________________________________________________________________________________ > Mario M. Moronne, Ph.D. > > [hidden email] > [hidden email] > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > |
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Thank you very much, I will you approach.
Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> > I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. Answer is yes. It is difficult or even impossible to detect nuclear antigens using such an intense fixation with PFA (4%, 40 min) followed by permeabilization with 0.025% triton. As a control you may also try 20 min fixation in methanol on ice (no triton is needed). Methanol fixation optionally may be followed by the immersion of cells in acetone for 10 sec and air dry. Methanol does not preserve the structure as good as PFA does, but it will reveal the distribution of your protein more efficiently. Art On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: > Mario, thank you very much for your response. > > I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. > My protein is a SEPT9 which is implicated in many cancerous transformations. > In both protocols FLAG and protein staining colocolize very well.. > Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. > I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. > Or, vice versa. My protocol does something strange that relocates my protein to nucleus. > > Thank you, > > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> > Olga, > > In and of itself, paraformaldehyde does not typically require Triton > as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % > Triton is still a fairly low concentration but maybe enough to punch > holes in your cell membranes causing a complete redistribution of > intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing > which protein you are tagging with FLAG, I can only speculate what > the consequences might be. > > It is a bit unclear what you mean by "opposite results." I am > assuming that in the first protocol (low Triton) you get what appears > to be colocalization of your protein and the FLAG tag, which is what > you logically would hope for. In the high Triton protocol, you lose > the peri-nuclear localization, but is this true also for the > protein-FLAG version, i.e., do the distributions remain coincident or > is that lost? If colocalization remains then one would be tempted to > blame the high Triton for possibly mobilizing the target proteins > away from the nucleus and into the cytoplasm. > > If target protein and the FLAG version no longer coincide then things > are more complicated. > > Anyway, using PF without detergent should work fine for fixation and > I would tend to believe it before accepting simultaneous PF and > Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? > > For sturdier fixation you might also try 3-4% PF plus 0.2% > glutaraldehyde in the initial fix. The PF goes in first and does the > initial fixation and allows glut. to follow and reinforce your target > crosslinking. > > With regards to using milk and BSA for blocking, I would seriously > consider changing over to an appropriate animal serum appropriate to > you antibodies, or better yet a product like Pierce's SuperBlock. > > Let us know how things go, > Mario > >>Hello everybody, >> >>This is my first message to confocal community. I am also molecular >>biologist:-(( and do not know all tricks in IF. >>I am trying to understand where is my FLAG tagged protein localized >>in breast epithelial cells which also has endogenous one. >>I grow cells in chamber slides. >>Old lab protocol show strong perinuclear staining for both ABs to >>our protein and FLAG. Protocol include: >>40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>for 1 hour. >> >>I was suspicious of too long paraformaldehyde treatment and too low >>Triton percentage. >> >>I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>get opposite result. >> >>In my variation our protein mostly cytoplasmic and nuclear. >> >>We also know that under some condition this protein create >>filamentous structure. >> >>If somebody could help me to understand which protocol is right, >>what kind of variation of protocol I could use to solve the problem, >>I would very much appreciate. >> >>Thank you, >> >>Olga >> >>Olga Makarova, PhD >>Research Specialist >>5323 MSRB III >> >> >>********************************************************** >>Electronic Mail is not secure, may not be read every day, and should >>not be used for urgent or sensitive issues > > > -- > ________________________________________________________________________________ > Mario M. Moronne, Ph.D. > > [hidden email] > [hidden email] > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling.
coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. Good luck Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > Thank you very much, I will you approach. > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. > > Answer is yes. It is difficult or even impossible to detect nuclear > antigens using such an intense fixation with PFA (4%, 40 min) followed > by permeabilization with 0.025% triton. > As a control you may also try 20 min fixation in methanol on ice (no > triton is needed). Methanol fixation optionally may be followed by the > immersion of cells in acetone for 10 sec and air dry. Methanol does > not preserve the structure as good as PFA does, but it will reveal the > distribution of your protein more efficiently. > Art > > On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: >> Mario, thank you very much for your response. >> >> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >> My protein is a SEPT9 which is implicated in many cancerous transformations. >> In both protocols FLAG and protein staining colocolize very well.. >> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >> >> Thank you, >> >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >> Olga, >> >> In and of itself, paraformaldehyde does not typically require Triton >> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >> Triton is still a fairly low concentration but maybe enough to punch >> holes in your cell membranes causing a complete redistribution of >> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >> which protein you are tagging with FLAG, I can only speculate what >> the consequences might be. >> >> It is a bit unclear what you mean by "opposite results." I am >> assuming that in the first protocol (low Triton) you get what appears >> to be colocalization of your protein and the FLAG tag, which is what >> you logically would hope for. In the high Triton protocol, you lose >> the peri-nuclear localization, but is this true also for the >> protein-FLAG version, i.e., do the distributions remain coincident or >> is that lost? If colocalization remains then one would be tempted to >> blame the high Triton for possibly mobilizing the target proteins >> away from the nucleus and into the cytoplasm. >> >> If target protein and the FLAG version no longer coincide then things >> are more complicated. >> >> Anyway, using PF without detergent should work fine for fixation and >> I would tend to believe it before accepting simultaneous PF and >> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >> >> For sturdier fixation you might also try 3-4% PF plus 0.2% >> glutaraldehyde in the initial fix. The PF goes in first and does the >> initial fixation and allows glut. to follow and reinforce your target >> crosslinking. >> >> With regards to using milk and BSA for blocking, I would seriously >> consider changing over to an appropriate animal serum appropriate to >> you antibodies, or better yet a product like Pierce's SuperBlock. >> >> Let us know how things go, >> Mario >> >>> Hello everybody, >>> >>> This is my first message to confocal community. I am also molecular >>> biologist:-(( and do not know all tricks in IF. >>> I am trying to understand where is my FLAG tagged protein localized >>> in breast epithelial cells which also has endogenous one. >>> I grow cells in chamber slides. >>> Old lab protocol show strong perinuclear staining for both ABs to >>> our protein and FLAG. Protocol include: >>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>> for 1 hour. >>> >>> I was suspicious of too long paraformaldehyde treatment and too low >>> Triton percentage. >>> >>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>> get opposite result. >>> >>> In my variation our protein mostly cytoplasmic and nuclear. >>> >>> We also know that under some condition this protein create >>> filamentous structure. >>> >>> If somebody could help me to understand which protocol is right, >>> what kind of variation of protocol I could use to solve the problem, >>> I would very much appreciate. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should >>> not be used for urgent or sensitive issues >> >> >> -- >> ________________________________________________________________________________ >> Mario M. Moronne, Ph.D. >> >> [hidden email] >> [hidden email] >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >> > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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Hi Everyone,
I could not agree more with Glen's comments. The more I see different immunofluorescent staining protocols (and the questions they raise), the more I realise that most researchers do not even question why they do a particular treatment. Fixation and permeabilisation techniques are good examples of "chinese whispers" where the technique gets handed from one person to the next without question. The choice of fixative (and the time and temperature of fixation) can be critical in determining if a particular antibody works at all. Formaldehyde (depolymerised paraformaldehyde) and methanol are commonly used - depending upon the location and shape of the epitope to be examined, they can give vastly different apparent affinities of the antibody. I think an important question that is almost never asked is "How long should I fix for?". I'm sure most people try a time suggested by someone else and, if they get some staining, never change or test their protocol further. Similarly, "what concentration should I use?" is frequently asked. I would highly recommend that everyone read the following article - I'm sure it will provide "food for thought". "Formaldehyde, Formalin, Paraformaldehyde and Glutaraldehyde: What They Are and What They Do." by John Kiernan, Microscopy Today, January, 2000, pages 8 - 12. ...rant finished :-) Cheers to all Paul Dr Paul Rigby Associate Professor Centre for Microscopy, Characterisation & Analysis (M510) The University of Western Australia 35 Stirling Highway Crawley WA 6009 Phone (61 8) 9346 2819 ________________________________ From: Confocal Microscopy List on behalf of Glen MacDonald Sent: Thu 20/05/2010 12:57 PM To: [hidden email] Subject: Re: Please help Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling. coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. Good luck Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > Thank you very much, I will you approach. > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. > > Answer is yes. It is difficult or even impossible to detect nuclear > antigens using such an intense fixation with PFA (4%, 40 min) followed > by permeabilization with 0.025% triton. > As a control you may also try 20 min fixation in methanol on ice (no > triton is needed). Methanol fixation optionally may be followed by the > immersion of cells in acetone for 10 sec and air dry. Methanol does > not preserve the structure as good as PFA does, but it will reveal the > distribution of your protein more efficiently. > Art > > On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: >> Mario, thank you very much for your response. >> >> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >> My protein is a SEPT9 which is implicated in many cancerous transformations. >> In both protocols FLAG and protein staining colocolize very well.. >> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >> >> Thank you, >> >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >> Olga, >> >> In and of itself, paraformaldehyde does not typically require Triton >> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >> Triton is still a fairly low concentration but maybe enough to punch >> holes in your cell membranes causing a complete redistribution of >> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >> which protein you are tagging with FLAG, I can only speculate what >> the consequences might be. >> >> It is a bit unclear what you mean by "opposite results." I am >> assuming that in the first protocol (low Triton) you get what appears >> to be colocalization of your protein and the FLAG tag, which is what >> you logically would hope for. In the high Triton protocol, you lose >> the peri-nuclear localization, but is this true also for the >> protein-FLAG version, i.e., do the distributions remain coincident or >> is that lost? If colocalization remains then one would be tempted to >> blame the high Triton for possibly mobilizing the target proteins >> away from the nucleus and into the cytoplasm. >> >> If target protein and the FLAG version no longer coincide then things >> are more complicated. >> >> Anyway, using PF without detergent should work fine for fixation and >> I would tend to believe it before accepting simultaneous PF and >> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >> >> For sturdier fixation you might also try 3-4% PF plus 0.2% >> glutaraldehyde in the initial fix. The PF goes in first and does the >> initial fixation and allows glut. to follow and reinforce your target >> crosslinking. >> >> With regards to using milk and BSA for blocking, I would seriously >> consider changing over to an appropriate animal serum appropriate to >> you antibodies, or better yet a product like Pierce's SuperBlock. >> >> Let us know how things go, >> Mario >> >>> Hello everybody, >>> >>> This is my first message to confocal community. I am also molecular >>> biologist:-(( and do not know all tricks in IF. >>> I am trying to understand where is my FLAG tagged protein localized >>> in breast epithelial cells which also has endogenous one. >>> I grow cells in chamber slides. >>> Old lab protocol show strong perinuclear staining for both ABs to >>> our protein and FLAG. Protocol include: >>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>> for 1 hour. >>> >>> I was suspicious of too long paraformaldehyde treatment and too low >>> Triton percentage. >>> >>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>> get opposite result. >>> >>> In my variation our protein mostly cytoplasmic and nuclear. >>> >>> We also know that under some condition this protein create >>> filamentous structure. >>> >>> If somebody could help me to understand which protocol is right, >>> what kind of variation of protocol I could use to solve the problem, >>> I would very much appreciate. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should >>> not be used for urgent or sensitive issues >> >> >> -- >> ________________________________________________________________________________ >> Mario M. Moronne, Ph.D. >> >> [hidden email] >> [hidden email] >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >> > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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To continue, whenever I tried a new antibody I would always do a
fixation and titration analysis ([fresh]PFA, GA, methanol, ethanol, nothing). Yes, it takes time and costs money in reagents, but at least you don't have to second guess that part of your assay from there forward. Sometimes you are surprised at the results. Certain antibodies work poorly with certain fixatives. Brian D Armstrong PhD Light Microscopy Core Manager Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag ing/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Rigby Sent: Thursday, May 20, 2010 10:45 AM To: [hidden email] Subject: Re: Please help Hi Everyone, I could not agree more with Glen's comments. The more I see different immunofluorescent staining protocols (and the questions they raise), the more I realise that most researchers do not even question why they do a particular treatment. Fixation and permeabilisation techniques are good examples of "chinese whispers" where the technique gets handed from one person to the next without question. The choice of fixative (and the time and temperature of fixation) can be critical in determining if a particular antibody works at all. Formaldehyde (depolymerised paraformaldehyde) and methanol are commonly used - depending upon the location and shape of the epitope to be examined, they can give vastly different apparent affinities of the antibody. I think an important question that is almost never asked is "How long should I fix for?". I'm sure most people try a time suggested by someone else and, if they get some staining, never change or test their protocol further. Similarly, "what concentration should I use?" is frequently asked. I would highly recommend that everyone read the following article - I'm sure it will provide "food for thought". "Formaldehyde, Formalin, Paraformaldehyde and Glutaraldehyde: What They Are and What They Do." by John Kiernan, Microscopy Today, January, 2000, pages 8 - 12. ...rant finished :-) Cheers to all Paul Dr Paul Rigby Associate Professor Centre for Microscopy, Characterisation & Analysis (M510) The University of Western Australia 35 Stirling Highway Crawley WA 6009 Phone (61 8) 9346 2819 ________________________________ From: Confocal Microscopy List on behalf of Glen MacDonald Sent: Thu 20/05/2010 12:57 PM To: [hidden email] Subject: Re: Please help Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling. coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. Good luck Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > Thank you very much, I will you approach. > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >> I guess , one of my questions is whether with the old protocol they and too little permeabilization with Triton. > > Answer is yes. It is difficult or even impossible to detect nuclear > antigens using such an intense fixation with PFA (4%, 40 min) followed > by permeabilization with 0.025% triton. > As a control you may also try 20 min fixation in methanol on ice (no > triton is needed). Methanol fixation optionally may be followed by the > immersion of cells in acetone for 10 sec and air dry. Methanol does > not preserve the structure as good as PFA does, but it will reveal the > distribution of your protein more efficiently. > Art > > On Thu, May 20, 2010 at 11:42 AM, Olga Makarova >> Mario, thank you very much for your response. >> >> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >> My protein is a SEPT9 which is implicated in many cancerous transformations. >> In both protocols FLAG and protein staining colocolize very well.. >> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >> >> Thank you, >> >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >> Olga, >> >> In and of itself, paraformaldehyde does not typically require Triton >> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >> Triton is still a fairly low concentration but maybe enough to punch >> holes in your cell membranes causing a complete redistribution of >> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >> which protein you are tagging with FLAG, I can only speculate what >> the consequences might be. >> >> It is a bit unclear what you mean by "opposite results." I am >> assuming that in the first protocol (low Triton) you get what appears >> to be colocalization of your protein and the FLAG tag, which is what >> you logically would hope for. In the high Triton protocol, you lose >> the peri-nuclear localization, but is this true also for the >> protein-FLAG version, i.e., do the distributions remain coincident or >> is that lost? If colocalization remains then one would be tempted to >> blame the high Triton for possibly mobilizing the target proteins >> away from the nucleus and into the cytoplasm. >> >> If target protein and the FLAG version no longer coincide then things >> are more complicated. >> >> Anyway, using PF without detergent should work fine for fixation and >> I would tend to believe it before accepting simultaneous PF and >> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >> >> For sturdier fixation you might also try 3-4% PF plus 0.2% >> glutaraldehyde in the initial fix. The PF goes in first and does the >> initial fixation and allows glut. to follow and reinforce your target >> crosslinking. >> >> With regards to using milk and BSA for blocking, I would seriously >> consider changing over to an appropriate animal serum appropriate to >> you antibodies, or better yet a product like Pierce's SuperBlock. >> >> Let us know how things go, >> Mario >> >>> Hello everybody, >>> >>> This is my first message to confocal community. I am also molecular >>> biologist:-(( and do not know all tricks in IF. >>> I am trying to understand where is my FLAG tagged protein localized >>> in breast epithelial cells which also has endogenous one. >>> I grow cells in chamber slides. >>> Old lab protocol show strong perinuclear staining for both ABs to >>> our protein and FLAG. Protocol include: >>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS >>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>> for 1 hour. >>> >>> I was suspicious of too long paraformaldehyde treatment and too low >>> Triton percentage. >>> >>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>> get opposite result. >>> >>> In my variation our protein mostly cytoplasmic and nuclear. >>> >>> We also know that under some condition this protein create >>> filamentous structure. >>> >>> If somebody could help me to understand which protocol is right, >>> what kind of variation of protocol I could use to solve the problem, >>> I would very much appreciate. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should >>> not be used for urgent or sensitive issues >> >> >> -- >> ________ >> Mario M. Moronne, Ph.D. >> >> [hidden email] >> [hidden email] >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >> > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues --------------------------------------------------------------------- SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). 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In reply to this post by Glen MacDonald-2
Hi Glen,
Thank you very much for your response. I am working with tissue culture cells. FLAG tagged ectopic protein and endogenous one are colocalized very well. I will definitely try to do only PFA, without Triton. However, everybody who responded, told we that the old protocol (4% PFA, 40min, then Triton 0,025% in every following blocking and washing solution) did not reveal nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5-20min) - all combination shows identical results - cytoplasmic and nuclear staining both for the Protein ABs and FLAG Cel Signalling general protocol also calls even for more Triton - like - 0.5%. They do not use ABs which do not work well in that general protocol. I was very happy about public consensus until your comments about Triton. Now a new question has come up: Could my protein redistributed to the nuclei because of Triton in my new protocol??? Which of these is TRUE?? Thank you, Olga Or, vice versa. My protocol does something strange that relocates my protein to nucleus. Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Glen MacDonald <[hidden email]> 5/20/2010 12:57 PM >>> Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling. coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. Good luck Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > Thank you very much, I will you approach. > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. > > Answer is yes. It is difficult or even impossible to detect nuclear > antigens using such an intense fixation with PFA (4%, 40 min) followed > by permeabilization with 0.025% triton. > As a control you may also try 20 min fixation in methanol on ice (no > triton is needed). Methanol fixation optionally may be followed by the > immersion of cells in acetone for 10 sec and air dry. Methanol does > not preserve the structure as good as PFA does, but it will reveal the > distribution of your protein more efficiently. > Art > > On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: >> Mario, thank you very much for your response. >> >> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >> My protein is a SEPT9 which is implicated in many cancerous transformations. >> In both protocols FLAG and protein staining colocolize very well.. >> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >> >> Thank you, >> >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >> Olga, >> >> In and of itself, paraformaldehyde does not typically require Triton >> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >> Triton is still a fairly low concentration but maybe enough to punch >> holes in your cell membranes causing a complete redistribution of >> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >> which protein you are tagging with FLAG, I can only speculate what >> the consequences might be. >> >> It is a bit unclear what you mean by "opposite results." I am >> assuming that in the first protocol (low Triton) you get what appears >> to be colocalization of your protein and the FLAG tag, which is what >> you logically would hope for. In the high Triton protocol, you lose >> the peri-nuclear localization, but is this true also for the >> protein-FLAG version, i.e., do the distributions remain coincident or >> is that lost? If colocalization remains then one would be tempted to >> blame the high Triton for possibly mobilizing the target proteins >> away from the nucleus and into the cytoplasm. >> >> If target protein and the FLAG version no longer coincide then things >> are more complicated. >> >> Anyway, using PF without detergent should work fine for fixation and >> I would tend to believe it before accepting simultaneous PF and >> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >> >> For sturdier fixation you might also try 3-4% PF plus 0.2% >> glutaraldehyde in the initial fix. The PF goes in first and does the >> initial fixation and allows glut. to follow and reinforce your target >> crosslinking. >> >> With regards to using milk and BSA for blocking, I would seriously >> consider changing over to an appropriate animal serum appropriate to >> you antibodies, or better yet a product like Pierce's SuperBlock. >> >> Let us know how things go, >> Mario >> >>> Hello everybody, >>> >>> This is my first message to confocal community. I am also molecular >>> biologist:-(( and do not know all tricks in IF. >>> I am trying to understand where is my FLAG tagged protein localized >>> in breast epithelial cells which also has endogenous one. >>> I grow cells in chamber slides. >>> Old lab protocol show strong perinuclear staining for both ABs to >>> our protein and FLAG. Protocol include: >>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>> for 1 hour. >>> >>> I was suspicious of too long paraformaldehyde treatment and too low >>> Triton percentage. >>> >>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>> get opposite result. >>> >>> In my variation our protein mostly cytoplasmic and nuclear. >>> >>> We also know that under some condition this protein create >>> filamentous structure. >>> >>> If somebody could help me to understand which protocol is right, >>> what kind of variation of protocol I could use to solve the problem, >>> I would very much appreciate. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should >>> not be used for urgent or sensitive issues >> >> >> -- >> ________________________________________________________________________________ >> Mario M. Moronne, Ph.D. >> >> [hidden email] >> [hidden email] >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >> > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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Olga,
Papers on location of nuclear antigens routinely use 10-15 min fixation with 2% formaldehyde, followed by ~5 min permeabilization in 0.25 - 0.5% triton x-100. Triton is used because it is non-ionic detergent and it does not destroy the cellular structure (in contrast ionic detergents like SDS can cause structural artifacts). Some people use other detergents e.g. saponin, but triton is most common. Upon fixation protein can not relocate to the cell nucleus by no means, however a shorter fixation and longer(stronger) permeabilization would help you to reveal the signal in the cell nucleus. The important difference in the protocols for location of nuclear and cytoplasmic antigens is that for the cytoplasmic antigens a weaker permeabilization is used. You should remember that If you increase a concentration of triton you may cause artifacts in the cytoplasm, so you may have to try several dilutions to optimize your protocol. On Thu, May 20, 2010 at 3:36 PM, Olga Makarova <[hidden email]> wrote: > Hi Glen, > > Thank you very much for your response. > I am working with tissue culture cells. > FLAG tagged ectopic protein and endogenous one are colocalized very well. > I will definitely try to do only PFA, without Triton. > However, everybody who responded, told we that the old protocol (4% PFA, 40min, then Triton 0,025% in every following blocking and washing solution) did not reveal nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. > My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5-20min) - all combination shows identical results - cytoplasmic and nuclear staining both for the Protein ABs and FLAG > Cel Signalling general protocol also calls even for more Triton - like - 0.5%. They do not use ABs which do not work well in that general protocol. > I was very happy about public consensus until your comments about Triton. > Now a new question has come up: Could my protein redistributed to the nuclei because of Triton in my new protocol??? > Which of these is TRUE?? > Thank you, > Olga > > Or, vice versa. My protocol does something strange that relocates my protein to nucleus. > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Glen MacDonald <[hidden email]> 5/20/2010 12:57 PM >>> > Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling. > > coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. > > Good luck > Glen > > Glen MacDonald > Core for Communication Research > Virginia Merrill Bloedel Hearing Research Center > Box 357923 > University of Washington > Seattle, WA 98195-7923 USA > (206) 616-4156 > [hidden email] > > > > > > > > > On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > >> Thank you very much, I will you approach. >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >>> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> >> Answer is yes. It is difficult or even impossible to detect nuclear >> antigens using such an intense fixation with PFA (4%, 40 min) followed >> by permeabilization with 0.025% triton. >> As a control you may also try 20 min fixation in methanol on ice (no >> triton is needed). Methanol fixation optionally may be followed by the >> immersion of cells in acetone for 10 sec and air dry. Methanol does >> not preserve the structure as good as PFA does, but it will reveal the >> distribution of your protein more efficiently. >> Art >> >> On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: >>> Mario, thank you very much for your response. >>> >>> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >>> My protein is a SEPT9 which is implicated in many cancerous transformations. >>> In both protocols FLAG and protein staining colocolize very well.. >>> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >>> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >>> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >>> Olga, >>> >>> In and of itself, paraformaldehyde does not typically require Triton >>> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >>> Triton is still a fairly low concentration but maybe enough to punch >>> holes in your cell membranes causing a complete redistribution of >>> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >>> which protein you are tagging with FLAG, I can only speculate what >>> the consequences might be. >>> >>> It is a bit unclear what you mean by "opposite results." I am >>> assuming that in the first protocol (low Triton) you get what appears >>> to be colocalization of your protein and the FLAG tag, which is what >>> you logically would hope for. In the high Triton protocol, you lose >>> the peri-nuclear localization, but is this true also for the >>> protein-FLAG version, i.e., do the distributions remain coincident or >>> is that lost? If colocalization remains then one would be tempted to >>> blame the high Triton for possibly mobilizing the target proteins >>> away from the nucleus and into the cytoplasm. >>> >>> If target protein and the FLAG version no longer coincide then things >>> are more complicated. >>> >>> Anyway, using PF without detergent should work fine for fixation and >>> I would tend to believe it before accepting simultaneous PF and >>> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >>> >>> For sturdier fixation you might also try 3-4% PF plus 0.2% >>> glutaraldehyde in the initial fix. The PF goes in first and does the >>> initial fixation and allows glut. to follow and reinforce your target >>> crosslinking. >>> >>> With regards to using milk and BSA for blocking, I would seriously >>> consider changing over to an appropriate animal serum appropriate to >>> you antibodies, or better yet a product like Pierce's SuperBlock. >>> >>> Let us know how things go, >>> Mario >>> >>>> Hello everybody, >>>> >>>> This is my first message to confocal community. I am also molecular >>>> biologist:-(( and do not know all tricks in IF. >>>> I am trying to understand where is my FLAG tagged protein localized >>>> in breast epithelial cells which also has endogenous one. >>>> I grow cells in chamber slides. >>>> Old lab protocol show strong perinuclear staining for both ABs to >>>> our protein and FLAG. Protocol include: >>>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>>> for 1 hour. >>>> >>>> I was suspicious of too long paraformaldehyde treatment and too low >>>> Triton percentage. >>>> >>>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>>> get opposite result. >>>> >>>> In my variation our protein mostly cytoplasmic and nuclear. >>>> >>>> We also know that under some condition this protein create >>>> filamentous structure. >>>> >>>> If somebody could help me to understand which protocol is right, >>>> what kind of variation of protocol I could use to solve the problem, >>>> I would very much appreciate. >>>> >>>> Thank you, >>>> >>>> Olga >>>> >>>> Olga Makarova, PhD >>>> Research Specialist >>>> 5323 MSRB III >>>> >>>> >>>> ********************************************************** >>>> Electronic Mail is not secure, may not be read every day, and should >>>> not be used for urgent or sensitive issues >>> >>> >>> -- >>> ________________________________________________________________________________ >>> Mario M. Moronne, Ph.D. >>> >>> [hidden email] >>> [hidden email] >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >>> >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > |
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In reply to this post by Olga Makarova
Dear Olga,
the article to which I referred: Melan and Sluder (1992) J. Cell Science. The question of rearrangement will depend upon how well the antigens are bound to other proteins and resistant to solubilizing. Triton can be very helpful in maintaining proteins in solution to prevent precipitation and non-specific binding. Some antibodies are more susceptible than others. In a polyclonal antisera if some antibodies are sticky its probably not so big a problem as with a monoclonal where such problems affect the entire population of molecules. How are you are labeling the proteins and observing them? If using fluorescence, I'd avoid glutaraldehyde due to its high fluorescence. But Mario is correct that is may help if you are using brightfield labels. > I am working with tissue culture cells. > FLAG tagged ectopic protein and endogenous one are colocalized very well. How was this determined? > I will definitely try to do only PFA, without Triton. Yes, and maybe also try 1% or 2% PFA, and try cold methanol as suggested. > However, everybody who responded, told we that the old protocol (4% PFA, 40min, then Triton 0,025% in every following blocking and washing solution) did not reveal nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. That is very possible, but without testing it is only an opinion consistent with "what everyone else does". > My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5-20min) - all combination shows identical results - cytoplasmic and nuclear staining both for the Protein ABs and FLAG > Cel Signalling general protocol also calls even for more Triton - like - 0.5%. They do not use ABs which do not work well in that general protocol. > I was very happy about public consensus until your comments about Triton. Most protocols from vendors are just as untested as any other published protocol. Mostly taken from the literature, sometimes developed in-house with tissues known to produce a robust signal. It worked - sweet, get some pictures!. And often biased towards robust signals with short incubation times using high concentrations of primary and secondary antibodies to make the product convenient to use. By the way, have you titered your antibody concentrations? What do you see with substituting blocking solution for the primary antibody at the current dilution of secondary? It is also possible that something else is affecting distribution of protein, such as culture conditions. Could the perinuclear distribution have been associated with organelles? Also, are you using the exact same culture conditions and antibodies as in your prior lab? Regards, Glen > Now a new question has come up: Could my protein redistributed to the nuclei because of Triton in my new protocol??? > Which of these is TRUE?? > Thank you, > Olga > > Or, vice versa. My protocol does something strange that relocates my protein to nucleus. > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Glen MacDonald <[hidden email]> 5/20/2010 12:57 PM >>> > Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling. > > coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. > > Good luck > Glen > > Glen MacDonald > Core for Communication Research > Virginia Merrill Bloedel Hearing Research Center > Box 357923 > University of Washington > Seattle, WA 98195-7923 USA > (206) 616-4156 > [hidden email] > > > > > > > > > On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > >> Thank you very much, I will you approach. >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >>> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> >> Answer is yes. It is difficult or even impossible to detect nuclear >> antigens using such an intense fixation with PFA (4%, 40 min) followed >> by permeabilization with 0.025% triton. >> As a control you may also try 20 min fixation in methanol on ice (no >> triton is needed). Methanol fixation optionally may be followed by the >> immersion of cells in acetone for 10 sec and air dry. Methanol does >> not preserve the structure as good as PFA does, but it will reveal the >> distribution of your protein more efficiently. >> Art >> >> On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: >>> Mario, thank you very much for your response. >>> >>> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >>> My protein is a SEPT9 which is implicated in many cancerous transformations. >>> In both protocols FLAG and protein staining colocolize very well.. >>> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >>> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >>> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >>> Olga, >>> >>> In and of itself, paraformaldehyde does not typically require Triton >>> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >>> Triton is still a fairly low concentration but maybe enough to punch >>> holes in your cell membranes causing a complete redistribution of >>> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >>> which protein you are tagging with FLAG, I can only speculate what >>> the consequences might be. >>> >>> It is a bit unclear what you mean by "opposite results." I am >>> assuming that in the first protocol (low Triton) you get what appears >>> to be colocalization of your protein and the FLAG tag, which is what >>> you logically would hope for. In the high Triton protocol, you lose >>> the peri-nuclear localization, but is this true also for the >>> protein-FLAG version, i.e., do the distributions remain coincident or >>> is that lost? If colocalization remains then one would be tempted to >>> blame the high Triton for possibly mobilizing the target proteins >>> away from the nucleus and into the cytoplasm. >>> >>> If target protein and the FLAG version no longer coincide then things >>> are more complicated. >>> >>> Anyway, using PF without detergent should work fine for fixation and >>> I would tend to believe it before accepting simultaneous PF and >>> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >>> >>> For sturdier fixation you might also try 3-4% PF plus 0.2% >>> glutaraldehyde in the initial fix. The PF goes in first and does the >>> initial fixation and allows glut. to follow and reinforce your target >>> crosslinking. >>> >>> With regards to using milk and BSA for blocking, I would seriously >>> consider changing over to an appropriate animal serum appropriate to >>> you antibodies, or better yet a product like Pierce's SuperBlock. >>> >>> Let us know how things go, >>> Mario >>> >>>> Hello everybody, >>>> >>>> This is my first message to confocal community. I am also molecular >>>> biologist:-(( and do not know all tricks in IF. >>>> I am trying to understand where is my FLAG tagged protein localized >>>> in breast epithelial cells which also has endogenous one. >>>> I grow cells in chamber slides. >>>> Old lab protocol show strong perinuclear staining for both ABs to >>>> our protein and FLAG. Protocol include: >>>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>>> for 1 hour. >>>> >>>> I was suspicious of too long paraformaldehyde treatment and too low >>>> Triton percentage. >>>> >>>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>>> get opposite result. >>>> >>>> In my variation our protein mostly cytoplasmic and nuclear. >>>> >>>> We also know that under some condition this protein create >>>> filamentous structure. >>>> >>>> If somebody could help me to understand which protocol is right, >>>> what kind of variation of protocol I could use to solve the problem, >>>> I would very much appreciate. >>>> >>>> Thank you, >>>> >>>> Olga >>>> >>>> Olga Makarova, PhD >>>> Research Specialist >>>> 5323 MSRB III >>>> >>>> >>>> ********************************************************** >>>> Electronic Mail is not secure, may not be read every day, and should >>>> not be used for urgent or sensitive issues >>> >>> >>> -- >>> ________________________________________________________________________________ >>> Mario M. Moronne, Ph.D. >>> >>> [hidden email] >>> [hidden email] >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >>> >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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Glen and others,
Thank you very very much to you and everybody who responded. I am going to read all suggested articles and do recommended experiments to be more prepared for discussion in the field where I am not an expert. My answers to your questions: Other scientists used cold methanol and observed nuclear staining. I used confocal and regular flourescent microscope to observe colocalization of my protein. I use mouse monoclonal anti-FLAG antibody and rabbit policlonal custom made affinity purified ABs to the part of the protein. One of the problems is my cells lines all have endogenous one. Secondary only staining showed no signal under my settings. Culture condition are the same in all experiments. I guess I am afraid to see again different signal in different condition without hope to know which one is true, unless I found an answer in suggested papers. It seems like that every result I will get could be explained by difference in methods. Thank a lot, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Glen MacDonald <[hidden email]> 5/20/2010 4:23 PM >>> Dear Olga, the article to which I referred: Melan and Sluder (1992) J. Cell Science. The question of rearrangement will depend upon how well the antigens are bound to other proteins and resistant to solubilizing. Triton can be very helpful in maintaining proteins in solution to prevent precipitation and non-specific binding. Some antibodies are more susceptible than others. In a polyclonal antisera if some antibodies are sticky its probably not so big a problem as with a monoclonal where such problems affect the entire population of molecules. How are you are labeling the proteins and observing them? If using fluorescence, I'd avoid glutaraldehyde due to its high fluorescence. But Mario is correct that is may help if you are using brightfield labels. > I am working with tissue culture cells. > FLAG tagged ectopic protein and endogenous one are colocalized very well. How was this determined? > I will definitely try to do only PFA, without Triton. Yes, and maybe also try 1% or 2% PFA, and try cold methanol as suggested. > However, everybody who responded, told we that the old protocol (4% PFA, 40min, then Triton 0,025% in every following blocking and washing solution) did not reveal nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. That is very possible, but without testing it is only an opinion consistent with "what everyone else does". > My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5-20min) - all combination shows identical results - cytoplasmic and nuclear staining both for the Protein ABs and FLAG > Cel Signalling general protocol also calls even for more Triton - like - 0.5%. They do not use ABs which do not work well in that general protocol. > I was very happy about public consensus until your comments about Triton. Most protocols from vendors are just as untested as any other published protocol. Mostly taken from the literature, sometimes developed in-house with tissues known to produce a robust signal. It worked - sweet, get some pictures!. And often biased towards robust signals with short incubation times using high concentrations of primary and secondary antibodies to make the product convenient to use. By the way, have you titered your antibody concentrations? What do you see with substituting blocking solution for the primary antibody at the current dilution of secondary? It is also possible that something else is affecting distribution of protein, such as culture conditions. Could the perinuclear distribution have been associated with organelles? Also, are you using the exact same culture conditions and antibodies as in your prior lab? Regards, Glen > Now a new question has come up: Could my protein redistributed to the nuclei because of Triton in my new protocol??? > Which of these is TRUE?? > Thank you, > Olga > > Or, vice versa. My protocol does something strange that relocates my protein to nucleus. > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > >>>> Glen MacDonald <[hidden email]> 5/20/2010 12:57 PM >>> > Generally, PFA sufficiently permeabilizes for most immunolabeling. PFA crosslinking may distort the antigen rather than block access. A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it. Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better. I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution. Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C. thaw slowly in the refrigerator and return to PBS. the ice crystals rip big holes in the membranes but the histology is much better than a frozen section. This might work with cultured cells. You could try shorter fixation, say 3 min. or 1% PFA. a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling. > > coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone. Carnoy's solution and fixes with picric acid are also options. > > Good luck > Glen > > Glen MacDonald > Core for Communication Research > Virginia Merrill Bloedel Hearing Research Center > Box 357923 > University of Washington > Seattle, WA 98195-7923 USA > (206) 616-4156 > [hidden email] > > > > > > > > > On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > >> Thank you very much, I will you approach. >> Olga >> >> Olga Makarova, PhD >> Research Specialist >> 5323 MSRB III >> >> >>>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >>> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >> >> Answer is yes. It is difficult or even impossible to detect nuclear >> antigens using such an intense fixation with PFA (4%, 40 min) followed >> by permeabilization with 0.025% triton. >> As a control you may also try 20 min fixation in methanol on ice (no >> triton is needed). Methanol fixation optionally may be followed by the >> immersion of cells in acetone for 10 sec and air dry. Methanol does >> not preserve the structure as good as PFA does, but it will reveal the >> distribution of your protein more efficiently. >> Art >> >> On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[hidden email]> wrote: >>> Mario, thank you very much for your response. >>> >>> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton. >>> My protein is a SEPT9 which is implicated in many cancerous transformations. >>> In both protocols FLAG and protein staining colocolize very well.. >>> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining. >>> I guess , one of my questions is whether with the old protocol they did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton. >>> Or, vice versa. My protocol does something strange that relocates my protein to nucleus. >>> >>> Thank you, >>> >>> Olga >>> >>> Olga Makarova, PhD >>> Research Specialist >>> 5323 MSRB III >>> >>> >>>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >>> Olga, >>> >>> In and of itself, paraformaldehyde does not typically require Triton >>> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 % >>> Triton is still a fairly low concentration but maybe enough to punch >>> holes in your cell membranes causing a complete redistribution of >>> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >>> which protein you are tagging with FLAG, I can only speculate what >>> the consequences might be. >>> >>> It is a bit unclear what you mean by "opposite results." I am >>> assuming that in the first protocol (low Triton) you get what appears >>> to be colocalization of your protein and the FLAG tag, which is what >>> you logically would hope for. In the high Triton protocol, you lose >>> the peri-nuclear localization, but is this true also for the >>> protein-FLAG version, i.e., do the distributions remain coincident or >>> is that lost? If colocalization remains then one would be tempted to >>> blame the high Triton for possibly mobilizing the target proteins >>> away from the nucleus and into the cytoplasm. >>> >>> If target protein and the FLAG version no longer coincide then things >>> are more complicated. >>> >>> Anyway, using PF without detergent should work fine for fixation and >>> I would tend to believe it before accepting simultaneous PF and >>> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton? >>> >>> For sturdier fixation you might also try 3-4% PF plus 0.2% >>> glutaraldehyde in the initial fix. The PF goes in first and does the >>> initial fixation and allows glut. to follow and reinforce your target >>> crosslinking. >>> >>> With regards to using milk and BSA for blocking, I would seriously >>> consider changing over to an appropriate animal serum appropriate to >>> you antibodies, or better yet a product like Pierce's SuperBlock. >>> >>> Let us know how things go, >>> Mario >>> >>>> Hello everybody, >>>> >>>> This is my first message to confocal community. I am also molecular >>>> biologist:-(( and do not know all tricks in IF. >>>> I am trying to understand where is my FLAG tagged protein localized >>>> in breast epithelial cells which also has endogenous one. >>>> I grow cells in chamber slides. >>>> Old lab protocol show strong perinuclear staining for both ABs to >>>> our protein and FLAG. Protocol include: >>>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT. >>>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton >>>> for 1 hour. >>>> >>>> I was suspicious of too long paraformaldehyde treatment and too low >>>> Triton percentage. >>>> >>>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and 20min and >>>> get opposite result. >>>> >>>> In my variation our protein mostly cytoplasmic and nuclear. >>>> >>>> We also know that under some condition this protein create >>>> filamentous structure. >>>> >>>> If somebody could help me to understand which protocol is right, >>>> what kind of variation of protocol I could use to solve the problem, >>>> I would very much appreciate. >>>> >>>> Thank you, >>>> >>>> Olga >>>> >>>> Olga Makarova, PhD >>>> Research Specialist >>>> 5323 MSRB III >>>> >>>> >>>> ********************************************************** >>>> Electronic Mail is not secure, may not be read every day, and should >>>> not be used for urgent or sensitive issues >>> >>> >>> -- >>> ________________________________________________________________________________ >>> Mario M. Moronne, Ph.D. >>> >>> [hidden email] >>> [hidden email] >>> ********************************************************** >>> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues >>> >> ********************************************************** >> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues |
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In reply to this post by Artem Pliss
Olga Saponin works as a “detergent” by forming a pore in the membrane at cholesterol. There are lots of cholesterol molecules in the plasma membrane and almost none in the nuclear envelope. Thus, using saponin will not allow antibodies to penetrate into the nucleus. If you use saponin for cytoplasmic antigens, include it in all rinse solutions as it washes out of the membrane if exposed to a rinse with no saponin. Dick Burry----- Original Message ----- From: Artem Pliss <[hidden email]> Date: Thursday, May 20, 2010 4:25 pm Subject: Re: Please help To: [hidden email] > Olga, > Papers on location of nuclear antigens routinely use 10-15 min > fixation with 2% formaldehyde, followed by ~5 min > permeabilization in > 0.25 - 0.5% triton x-100. Triton is used because it is non-ionic > detergent and it does not destroy the cellular structure > (in contrast > ionic detergents like SDS can cause structural artifacts). Some > people use other detergents e.g. saponin, but triton is most common. > Upon fixation protein can not relocate to the cell nucleus by no > means, however a shorter fixation and longer(stronger) > permeabilization would help you to reveal the signal in the cell > nucleus. The important difference in the protocols for location of > nuclear and cytoplasmic antigens is that for the cytoplasmic antigens > a weaker permeabilization is used. You should remember that If you > increase a concentration of triton you may cause artifacts in the > cytoplasm, so you may have to try several dilutions to optimize your > protocol. > > > > On Thu, May 20, 2010 at 3:36 PM, Olga Makarova > <[hidden email]> wrote: > > Hi Glen, > > > > Thank you very much for your response. > > I am working with tissue culture cells. > > FLAG tagged ectopic protein and endogenous one are colocalized > very well. > > I will definitely try to do only PFA, without Triton. > > However, everybody who responded, told we that the old > protocol (4% PFA, 40min, then Triton 0,025% in every following > blocking and washing solution) did not reveal nuclear staining > because of too much crosslinking by PFA and too little > permeabilization with Triton. > > My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5- > 20min) - all combination shows identical results - cytoplasmic > and nuclear staining both for the Protein ABs and FLAG > > Cel Signalling general protocol also calls even for more > Triton - like - 0.5%. They do not use ABs which do not work > well in that general protocol. > > I was very happy about public consensus until your comments > about Triton. > > Now a new question has come up: Could my protein > redistributed to the nuclei because of Triton in my new protocol??? > > Which of these is TRUE?? > > Thank you, > > Olga > > > > Or, vice versa. My protocol does something strange that > relocates my protein to nucleus. > > > > Olga Makarova, PhD > > Research Specialist > > 5323 MSRB III > > > > > >>>> Glen MacDonald <[hidden email]> 5/20/2010 > 12:57 PM >>> > > Generally, PFA sufficiently permeabilizes for most > immunolabeling. PFA crosslinking may distort the antigen rather > than block access. A lot of protocols include Triton because > they were inherited from someone else or that is just how > someone learned to do it. Many researchers never take the time > to see if it can be eliminated or reduced to achieve results > that are the same or better. I don't have the reference at > hand, but Triton has been shown to rearrange antigen > distribution. Other approaches with PFA tissue is to pass > vibratome sections through graded sucrose into 30% sucrose then > freeze at -80C. thaw slowly in the refrigerator and return to > PBS. the ice crystals rip big holes in the membranes but the > histology is much better than a frozen section. This might work > with cultured cells. You could try shorter fixation, say 3 min. > or 1% PFA. a 3 min treatment (10 min for 40 um vibratome > sections) with 1% SDS (an optimal concentration may be less) > before the blocking step may also improve the labeling. > > > > coagulating fixatives tend to preserve antigenicity better, > such as cold methanol or acetone. Carnoy's solution and fixes > with picric acid are also options. > > > > Good luck > > Glen > > > > Glen MacDonald > > Core for Communication Research > > Virginia Merrill Bloedel Hearing Research Center > > Box 357923 > > University of Washington > > Seattle, WA 98195-7923 USA > > (206) 616-4156 > > [hidden email] > > > > > > > > > > > > > > > > > > On May 20, 2010, at 9:31 AM, Olga Makarova wrote: > > > >> Thank you very much, I will you approach. > >> Olga > >> > >> Olga Makarova, PhD > >> Research Specialist > >> 5323 MSRB III > >> > >> > >>>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> > >>> I guess , one of my questions is whether with the old > protocol they did not see the nuclear staining because of too > much crosslinking by PFA and too little permeabilization with Triton. > >> > >> Answer is yes. It is difficult or even impossible to detect nuclear > >> antigens using such an intense fixation with PFA (4%, 40 min) > followed>> by permeabilization with 0.025% triton. > >> As a control you may also try 20 min fixation in methanol on > ice (no > >> triton is needed). Methanol fixation optionally may be > followed by the > >> immersion of cells in acetone for 10 sec and air dry. > Methanol does > >> not preserve the structure as good as PFA does, but it will > reveal the > >> distribution of your protein more efficiently. > >> Art > >> > >> On Thu, May 20, 2010 at 11:42 AM, Olga Makarova > <[hidden email]> wrote: > >>> Mario, thank you very much for your response. > >>> > >>> I am really confused about many things. I 've found many > protocols for IF and all of them ask for both the PFA and Triton. > >>> My protein is a SEPT9 which is implicated in many cancerous > transformations.>>> In both protocols FLAG and protein staining > colocolize very well.. > >>> Sorry for the "opposite" I meant that instead of familiar > perinuclear staining which was observed by people before, I see > nuclear and cytoplasmic staining. > >>> I guess , one of my questions is whether with the old > protocol they did not see the nuclear staining because of too > much crosslinking by PFA and too little permeabilization with Triton. > >>> Or, vice versa. My protocol does something strange that > relocates my protein to nucleus. > >>> > >>> Thank you, > >>> > >>> Olga > >>> > >>> Olga Makarova, PhD > >>> Research Specialist > >>> 5323 MSRB III > >>> > >>> > >>>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> > >>> Olga, > >>> > >>> In and of itself, paraformaldehyde does not typically > require Triton > >>> as it is quite membrane permeable unlike glutaraldehyde. > Adding 0.1 % > >>> Triton is still a fairly low concentration but maybe enough > to punch > >>> holes in your cell membranes causing a complete > redistribution of > >>> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing > >>> which protein you are tagging with FLAG, I can only > speculate what > >>> the consequences might be. > >>> > >>> It is a bit unclear what you mean by "opposite results." I am > >>> assuming that in the first protocol (low Triton) you get > what appears > >>> to be colocalization of your protein and the FLAG tag, which > is what > >>> you logically would hope for. In the high Triton protocol, > you lose > >>> the peri-nuclear localization, but is this true also for the > >>> protein-FLAG version, i.e., do the distributions remain > coincident or > >>> is that lost? If colocalization remains then one would be > tempted to > >>> blame the high Triton for possibly mobilizing the target proteins > >>> away from the nucleus and into the cytoplasm. > >>> > >>> If target protein and the FLAG version no longer coincide > then things > >>> are more complicated. > >>> > >>> Anyway, using PF without detergent should work fine for > fixation and > >>> I would tend to believe it before accepting simultaneous PF and > >>> Triton. Did you try the PF for 40 min. then the higher 0.1 % > Triton?>>> > >>> For sturdier fixation you might also try 3-4% PF plus 0.2% > >>> glutaraldehyde in the initial fix. The PF goes in first and > does the > >>> initial fixation and allows glut. to follow and reinforce > your target > >>> crosslinking. > >>> > >>> With regards to using milk and BSA for blocking, I would seriously > >>> consider changing over to an appropriate animal serum > appropriate to > >>> you antibodies, or better yet a product like Pierce's SuperBlock. > >>> > >>> Let us know how things go, > >>> Mario > >>> > >>>> Hello everybody, > >>>> > >>>> This is my first message to confocal community. I am also > molecular>>>> biologist:-(( and do not know all tricks in IF. > >>>> I am trying to understand where is my FLAG tagged protein > localized>>>> in breast epithelial cells which also has > endogenous one. > >>>> I grow cells in chamber slides. > >>>> Old lab protocol show strong perinuclear staining for both > ABs to > >>>> our protein and FLAG. Protocol include: > >>>> 40 min of 4% paraformaldehyde treatment (previously > frozen!!) in PBS at RT. > >>>> Permeabilization and blocking solution: 5%milk, 1% BSA, > 0.025%Triton>>>> for 1 hour. > >>>> > >>>> I was suspicious of too long paraformaldehyde treatment and > too low > >>>> Triton percentage. > >>>> > >>>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and > 20min and > >>>> get opposite result. > >>>> > >>>> In my variation our protein mostly cytoplasmic and nuclear. > >>>> > >>>> We also know that under some condition this protein create > >>>> filamentous structure. > >>>> > >>>> If somebody could help me to understand which protocol is right, > >>>> what kind of variation of protocol I could use to solve the > problem,>>>> I would very much appreciate. > >>>> > >>>> Thank you, > >>>> > >>>> Olga > >>>> > >>>> Olga Makarova, PhD > >>>> Research Specialist > >>>> 5323 MSRB III > >>>> > >>>> > >>>> ********************************************************** > >>>> Electronic Mail is not secure, may not be read every day, > and should > >>>> not be used for urgent or sensitive issues > >>> > >>> > >>> -- > >>> > ________________________________________________________________________________>>> Mario M. Moronne, Ph.D. > >>> > >>> [hidden email] > >>> [hidden email] > >>> ********************************************************** > >>> Electronic Mail is not secure, may not be read every day, > and should not be used for urgent or sensitive issues > >>> > >> ********************************************************** > >> Electronic Mail is not secure, may not be read every day, and > should not be used for urgent or sensitive issues > > ********************************************************** > > Electronic Mail is not secure, may not be read every day, and > should not be used for urgent or sensitive issues > > > > > -- > BEGIN-ANTISPAM-VOTING-LINKS > ------------------------------------------------------ > > Teach CanIt if this mail (ID 1041599261) is spam: > Spam: > https://antispam.osu.edu/b.php?i=1041599261&m=fff700ead800&c=sNot spam: https://antispam.osu.edu/b.php?i=1041599261&m=fff700ead800&c=n > Forget vote: > https://antispam.osu.edu/b.php?i=1041599261&m=fff700ead800&c=f--- > --------------------------------------------------- > END-ANTISPAM-VOTING-LINKS > |
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In reply to this post by Artem Pliss
Dear List members,
By coincidence a microscope user just walked into my office asking about methanol fixation. They fix cultured cells in cold methanol and leave the samples for up to two weeks in the methanol. This is not how I would prepare my material but what could be the consequences? Would extraction continue to happen or would you not expect much difference with 5 minutes cold methanol? Thanks Kees Dr K.R. Straatman Senior Experimental Officer College of Medicine, Biological Sciences and Psychology University of Leicester http://www.le.ac.uk/biochem/microscopy/home.html |
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I believe methanol depolymerizes actin (so no phalloidin staining, if
that matters). Otherwise, we've stored our immunohistochemistry material (mostly whole micometazoans and embryos) in cold methanol with good results, depending on the antibody. Julian Straatman, Kees R. (Dr.) wrote: > Dear List members, > > By coincidence a microscope user just walked into my office asking about methanol fixation. They fix cultured cells in cold methanol and leave the samples for up to two weeks in the methanol. This is not how I would prepare my material but what could be the consequences? Would extraction continue to happen or would you not expect much difference with 5 minutes cold methanol? > > Thanks > > Kees > > > Dr K.R. Straatman > Senior Experimental Officer > College of Medicine, Biological Sciences and Psychology > University of Leicester > > http://www.le.ac.uk/biochem/microscopy/home.html > > -- Julian P.S. Smith III Director, Winthrop Microscopy Facility Dept. of Biology Winthrop University 520 Cherry Rd. Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax) 803-524-2347 (cell) |
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ok, acuerdate de mandarme lo de los contratos de mantenimiento
Julian Smith III escribió: > I believe methanol depolymerizes actin (so no phalloidin staining, if > that matters). Otherwise, we've stored our immunohistochemistry > material (mostly whole micometazoans and embryos) in cold methanol > with good results, depending on the antibody. > Julian > > Straatman, Kees R. (Dr.) wrote: >> Dear List members, >> >> By coincidence a microscope user just walked into my office asking >> about methanol fixation. They fix cultured cells in cold methanol and >> leave the samples for up to two weeks in the methanol. This is not >> how I would prepare my material but what could be the consequences? >> Would extraction continue to happen or would you not expect much >> difference with 5 minutes cold methanol? >> Thanks >> >> Kees >> >> >> Dr K.R. Straatman >> Senior Experimental Officer >> College of Medicine, Biological Sciences and Psychology University of >> Leicester >> >> http://www.le.ac.uk/biochem/microscopy/home.html >> >> > > -- Juan Luis Ribas Servicio de Microscopía Centro de Investigación, Tecnología e Innovación Universidad de Sevilla Av. Reina Mercedes 4b 41012 Sevilla Tfno: 954559983 |
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In reply to this post by Julian Smith III
it will also quench any GFP (or similar) signal, but it wil be recoverable by using an anti-gfp antibody.
Cheers Cam ________________________________ From: Confocal Microscopy List on behalf of Julian Smith III Sent: Fri 21/05/2010 10:15 PM To: [hidden email] Subject: Re: more fixation I believe methanol depolymerizes actin (so no phalloidin staining, if that matters). Otherwise, we've stored our immunohistochemistry material (mostly whole micometazoans and embryos) in cold methanol with good results, depending on the antibody. Julian Straatman, Kees R. (Dr.) wrote: > Dear List members, > > By coincidence a microscope user just walked into my office asking about methanol fixation. They fix cultured cells in cold methanol and leave the samples for up to two weeks in the methanol. This is not how I would prepare my material but what could be the consequences? Would extraction continue to happen or would you not expect much difference with 5 minutes cold methanol? > > Thanks > > Kees > > > Dr K.R. Straatman > Senior Experimental Officer > College of Medicine, Biological Sciences and Psychology > University of Leicester > > http://www.le.ac.uk/biochem/microscopy/home.html > > -- Julian P.S. Smith III Director, Winthrop Microscopy Facility Dept. of Biology Winthrop University 520 Cherry Rd. Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax) 803-524-2347 (cell) |
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In reply to this post by RICHARD BURRY
Saponin is mostly used for the cytoplasm however at higher
concentrations in some protocols it can be used for the nuclear staining. I guess it depends on the properties of antibody. Below is a reference http://www.nature.com/cdd/journal/v11/n12/full/4401512a.html#fig1 However I think that for purposes of Olga she would do fine with either triton or methanol. On Thu, May 20, 2010 at 7:00 PM, RICHARD BURRY <[hidden email]> wrote: > Olga > > Saponin works as a “detergent” by forming a pore in the membrane at > cholesterol. There are lots of cholesterol molecules in the plasma membrane > and almost none in the nuclear envelope. Thus, using saponin will not allow > antibodies to penetrate into the nucleus. If you use saponin for > cytoplasmic antigens, include it in all rinse solutions as it washes out of > the membrane if exposed to a rinse with no saponin. > > Dick Burry > > ----- Original Message ----- > From: Artem Pliss <[hidden email]> > Date: Thursday, May 20, 2010 4:25 pm > Subject: Re: Please help > To: [hidden email] > >> Olga, >> Papers on location of nuclear antigens routinely use 10-15 min >> fixation with 2% formaldehyde, followed by ~5 min >> permeabilization in >> 0.25 - 0.5% triton x-100. Triton is used because it is non-ionic >> detergent and it does not destroy the cellular structure >> (in contrast >> ionic detergents like SDS can cause structural artifacts). Some >> people use other detergents e.g. saponin, but triton is most common. >> Upon fixation protein can not relocate to the cell nucleus by no >> means, however a shorter fixation and longer(stronger) >> permeabilization would help you to reveal the signal in the cell >> nucleus. The important difference in the protocols for location of >> nuclear and cytoplasmic antigens is that for the cytoplasmic antigens >> a weaker permeabilization is used. You should remember that If you >> increase a concentration of triton you may cause artifacts in the >> cytoplasm, so you may have to try several dilutions to optimize your >> protocol. >> >> >> >> On Thu, May 20, 2010 at 3:36 PM, Olga Makarova >> <[hidden email]> wrote: >> > Hi Glen, >> > >> > Thank you very much for your response. >> > I am working with tissue culture cells. >> > FLAG tagged ectopic protein and endogenous one are colocalized >> very well. >> > I will definitely try to do only PFA, without Triton. >> > However, everybody who responded, told we that the old >> protocol (4% PFA, 40min, then Triton 0,025% in every following >> blocking and washing solution) did not reveal nuclear staining >> because of too much crosslinking by PFA and too little >> permeabilization with Triton. >> > My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5- >> 20min) - all combination shows identical results - cytoplasmic >> and nuclear staining both for the Protein ABs and FLAG >> > Cel Signalling general protocol also calls even for more >> Triton - like - 0.5%. They do not use ABs which do not work >> well in that general protocol. >> > I was very happy about public consensus until your comments >> about Triton. >> > Now a new question has come up: Could my protein >> redistributed to the nuclei because of Triton in my new protocol??? >> > Which of these is TRUE?? >> > Thank you, >> > Olga >> > >> > Or, vice versa. My protocol does something strange that >> relocates my protein to nucleus. >> > >> > Olga Makarova, PhD >> > Research Specialist >> > 5323 MSRB III >> > >> > >> >>>> Glen MacDonald <[hidden email]> 5/20/2010 >> 12:57 PM >>> >> > Generally, PFA sufficiently permeabilizes for most >> immunolabeling. PFA crosslinking may distort the antigen rather >> than block access. A lot of protocols include Triton because >> they were inherited from someone else or that is just how >> someone learned to do it. Many researchers never take the time >> to see if it can be eliminated or reduced to achieve results >> that are the same or better. I don't have the reference at >> hand, but Triton has been shown to rearrange antigen >> distribution. Other approaches with PFA tissue is to pass >> vibratome sections through graded sucrose into 30% sucrose then >> freeze at -80C. thaw slowly in the refrigerator and return to >> PBS. the ice crystals rip big holes in the membranes but the >> histology is much better than a frozen section. This might work >> with cultured cells. You could try shorter fixation, say 3 min. >> or 1% PFA. a 3 min treatment (10 min for 40 um vibratome >> sections) with 1% SDS (an optimal concentration may be less) >> before the blocking step may also improve the labeling. >> > >> > coagulating fixatives tend to preserve antigenicity better, >> such as cold methanol or acetone. Carnoy's solution and fixes >> with picric acid are also options. >> > >> > Good luck >> > Glen >> > >> > Glen MacDonald >> > Core for Communication Research >> > Virginia Merrill Bloedel Hearing Research Center >> > Box 357923 >> > University of Washington >> > Seattle, WA 98195-7923 USA >> > (206) 616-4156 >> > [hidden email] >> > >> > >> > >> > >> > >> > >> > >> > >> > On May 20, 2010, at 9:31 AM, Olga Makarova wrote: >> > >> >> Thank you very much, I will you approach. >> >> Olga >> >> >> >> Olga Makarova, PhD >> >> Research Specialist >> >> 5323 MSRB III >> >> >> >> >> >>>>> Artem Pliss <[hidden email]> 5/20/2010 12:16 PM >>> >> >>> I guess , one of my questions is whether with the old >> protocol they did not see the nuclear staining because of too >> much crosslinking by PFA and too little permeabilization with Triton. >> >> >> >> Answer is yes. It is difficult or even impossible to detect nuclear >> >> antigens using such an intense fixation with PFA (4%, 40 min) >> followed>> by permeabilization with 0.025% triton. >> >> As a control you may also try 20 min fixation in methanol on >> ice (no >> >> triton is needed). Methanol fixation optionally may be >> followed by the >> >> immersion of cells in acetone for 10 sec and air dry. >> Methanol does >> >> not preserve the structure as good as PFA does, but it will >> reveal the >> >> distribution of your protein more efficiently. >> >> Art >> >> >> >> On Thu, May 20, 2010 at 11:42 AM, Olga Makarova >> <[hidden email]> wrote: >> >>> Mario, thank you very much for your response. >> >>> >> >>> I am really confused about many things. I 've found many >> protocols for IF and all of them ask for both the PFA and Triton. >> >>> My protein is a SEPT9 which is implicated in many cancerous >> transformations.>>> In both protocols FLAG and protein staining >> colocolize very well.. >> >>> Sorry for the "opposite" I meant that instead of familiar >> perinuclear staining which was observed by people before, I see >> nuclear and cytoplasmic staining. >> >>> I guess , one of my questions is whether with the old >> protocol they did not see the nuclear staining because of too >> much crosslinking by PFA and too little permeabilization with Triton. >> >>> Or, vice versa. My protocol does something strange that >> relocates my protein to nucleus. >> >>> >> >>> Thank you, >> >>> >> >>> Olga >> >>> >> >>> Olga Makarova, PhD >> >>> Research Specialist >> >>> 5323 MSRB III >> >>> >> >>> >> >>>>>> Mario <[hidden email]> 5/19/2010 3:01 PM >>> >> >>> Olga, >> >>> >> >>> In and of itself, paraformaldehyde does not typically >> require Triton >> >>> as it is quite membrane permeable unlike glutaraldehyde. >> Adding 0.1 % >> >>> Triton is still a fairly low concentration but maybe enough >> to punch >> >>> holes in your cell membranes causing a complete >> redistribution of >> >>> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing >> >>> which protein you are tagging with FLAG, I can only >> speculate what >> >>> the consequences might be. >> >>> >> >>> It is a bit unclear what you mean by "opposite results." I am >> >>> assuming that in the first protocol (low Triton) you get >> what appears >> >>> to be colocalization of your protein and the FLAG tag, which >> is what >> >>> you logically would hope for. In the high Triton protocol, >> you lose >> >>> the peri-nuclear localization, but is this true also for the >> >>> protein-FLAG version, i.e., do the distributions remain >> coincident or >> >>> is that lost? If colocalization remains then one would be >> tempted to >> >>> blame the high Triton for possibly mobilizing the target proteins >> >>> away from the nucleus and into the cytoplasm. >> >>> >> >>> If target protein and the FLAG version no longer coincide >> then things >> >>> are more complicated. >> >>> >> >>> Anyway, using PF without detergent should work fine for >> fixation and >> >>> I would tend to believe it before accepting simultaneous PF and >> >>> Triton. Did you try the PF for 40 min. then the higher 0.1 % >> Triton?>>> >> >>> For sturdier fixation you might also try 3-4% PF plus 0.2% >> >>> glutaraldehyde in the initial fix. The PF goes in first and >> does the >> >>> initial fixation and allows glut. to follow and reinforce >> your target >> >>> crosslinking. >> >>> >> >>> With regards to using milk and BSA for blocking, I would seriously >> >>> consider changing over to an appropriate animal serum >> appropriate to >> >>> you antibodies, or better yet a product like Pierce's SuperBlock. >> >>> >> >>> Let us know how things go, >> >>> Mario >> >>> >> >>>> Hello everybody, >> >>>> >> >>>> This is my first message to confocal community. I am also >> molecular>>>> biologist:-(( and do not know all tricks in IF. >> >>>> I am trying to understand where is my FLAG tagged protein >> localized>>>> in breast epithelial cells which also has >> endogenous one. >> >>>> I grow cells in chamber slides. >> >>>> Old lab protocol show strong perinuclear staining for both >> ABs to >> >>>> our protein and FLAG. Protocol include: >> >>>> 40 min of 4% paraformaldehyde treatment (previously >> frozen!!) in PBS at RT. >> >>>> Permeabilization and blocking solution: 5%milk, 1% BSA, >> 0.025%Triton>>>> for 1 hour. >> >>>> >> >>>> I was suspicious of too long paraformaldehyde treatment and >> too low >> >>>> Triton percentage. >> >>>> >> >>>> I tried fresh PF 5 and 20 min with Triton 0.1% for 5 and >> 20min and >> >>>> get opposite result. >> >>>> >> >>>> In my variation our protein mostly cytoplasmic and nuclear. >> >>>> >> >>>> We also know that under some condition this protein create >> >>>> filamentous structure. >> >>>> >> >>>> If somebody could help me to understand which protocol is right, >> >>>> what kind of variation of protocol I could use to solve the >> problem,>>>> I would very much appreciate. >> >>>> >> >>>> Thank you, >> >>>> >> >>>> Olga >> >>>> >> >>>> Olga Makarova, PhD >> >>>> Research Specialist >> >>>> 5323 MSRB III >> >>>> >> >>>> >> >>>> ********************************************************** >> >>>> Electronic Mail is not secure, may not be read every day, >> and should >> >>>> not be used for urgent or sensitive issues >> >>> >> >>> >> >>> -- >> >>> >> >> ________________________________________________________________________________>>> >> Mario M. Moronne, Ph.D. >> >>> >> >>> [hidden email] >> >>> [hidden email] >> >>> ********************************************************** >> >>> Electronic Mail is not secure, may not be read every day, >> and should not be used for urgent or sensitive issues >> >>> >> >> ********************************************************** >> >> Electronic Mail is not secure, may not be read every day, and >> should not be used for urgent or sensitive issues >> > ********************************************************** >> > Electronic Mail is not secure, may not be read every day, and >> should not be used for urgent or sensitive issues >> > >> >> >> -- >> BEGIN-ANTISPAM-VOTING-LINKS >> ------------------------------------------------------ >> >> Teach CanIt if this mail (ID 1041599261) is spam: >> Spam: >> https://antispam.osu.edu/b.php?i=1041599261&m=fff700ead800&c=sNot spam: >> https://antispam.osu.edu/b.php?i=1041599261&m=fff700ead800&c=n >> Forget vote: >> https://antispam.osu.edu/b.php?i=1041599261&m=fff700ead800&c=f--- >> --------------------------------------------------- >> END-ANTISPAM-VOTING-LINKS >> > |
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In reply to this post by Cameron Nowell
Hi All,
I would be very interested to find out what success people with methanol fixation. I have heard many people suggest that a coagulative fixative (like methanol) is better than paraformaldehyde for preserving cytoskeletal elements. Is this everyone's experience? Also, many years ago, cold fixation (with methanol or paraformaldehyde) was reported to cause depolymerisation of microtubules (so you had to fix at room temperature to see microtubular filaments). Is this still the generally accepted view? Any comments from personal experience? Thanks everyone Paul Dr Paul Rigby Associate Professor Centre for Microscopy, Characterisation & Analysis (M510) The University of Western Australia 35 Stirling Highway Crawley WA 6009 Phone (61 8) 9346 2819 ________________________________ From: Confocal Microscopy List on behalf of Cameron Nowell Sent: Fri 21/05/2010 8:25 AM To: [hidden email] Subject: Re: more fixation it will also quench any GFP (or similar) signal, but it wil be recoverable by using an anti-gfp antibody. Cheers Cam ________________________________ From: Confocal Microscopy List on behalf of Julian Smith III Sent: Fri 21/05/2010 10:15 PM To: [hidden email] Subject: Re: more fixation I believe methanol depolymerizes actin (so no phalloidin staining, if that matters). Otherwise, we've stored our immunohistochemistry material (mostly whole micometazoans and embryos) in cold methanol with good results, depending on the antibody. Julian Straatman, Kees R. (Dr.) wrote: > Dear List members, > > By coincidence a microscope user just walked into my office asking about methanol fixation. They fix cultured cells in cold methanol and leave the samples for up to two weeks in the methanol. This is not how I would prepare my material but what could be the consequences? Would extraction continue to happen or would you not expect much difference with 5 minutes cold methanol? > > Thanks > > Kees > > Dr K.R. Straatman > Senior Experimental Officer > College of Medicine, Biological Sciences and Psychology > University of Leicester > > http://www.le.ac.uk/biochem/microscopy/home.html > Julian P.S. Smith III Director, Winthrop Microscopy Facility Dept. of Biology Winthrop University 520 Cherry Rd. Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax) 803-524-2347 (cell) |
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