Please help
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Hello all;
Great discussions on this listserve. For kicks one time, I tried many different fixations while trying to fluorescence immunoprobe for a known antigen, but in a new system. I tried 4% PFA, 2% PFA, ice-cold dry methanol, ethanol/acetic acid, picric acid, or 1% glutaraldehyde, all in recommended times and temperatures. With my one antibody and my one cell culture system, I obtained 6 different localizations, some drastically unique. Which one was real? Further investigation required, but these disparate results clued us in to novel ideas. It was a good exercise in antigen characterization. By comparing the results with the chemistry involved in how each fixative actually works, we came up with some good and interesting results. Lesson learned was that there is no ONE fixative for everything. Try a bunch, and see what you get. It may be initially frustrating, but it may be very revealing in the end. BTW Paul (good to hear from you again!), when working on intermediate filaments, we fixed in ice-cold DRY methanol (use Molecular Sieve) for 2 min. This was our routine fixation. Only looked at microtubules once with this fix, and it worked well, but we weren't being quantitative about this analysis. Kathy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paul Rigby Sent: Friday, May 21, 2010 7:48 AM To: [hidden email] Subject: Re: more fixation 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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In reply to this post by Artem Pliss
Hello,
Thank you all for your responses. After reading recommended literature : Melan and Sluder and many other papers (may be because of lucking some basic knowledge in cell biology) I realized that I have to try many different protocol and their variations. However, I know that I will get very different results, all of which could be artifactual partially, because of redistribution after Triton permeabilization including artificial nuclear location, or artificial nuclear exclusion. My protein (75kD) is pretty much new, so we do not know its function and localization. I work only with attached epithelial cells lines. I would be very grateful if somebody could direct me to the simplest string of experiments (unfortunately, I do not have a lots of time) and may be willing to analyze the results. Thank you very much for your time, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Artem Pliss <[hidden email]> 5/21/2010 10:32 AM >>> 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 >> 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 >> >> >> >> 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 >> >>> 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 >> >>> 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 >> >>> 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 >> >>> 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 >> >>>> 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 >> 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 >> > ********************************************************** 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 Mario-2
Hello,
not a confocal question, but I hope someone may enlighten me. One of our users needs to replace the lamp housing on her Zeiss fluorescence microscope. The Zeiss rep recommends switching to a HXP lamp and housing. She needs to know that the HXP lamp will give equal or better excitation than HBO of especially Alexa 488 and Alexa 546. I have no experience of HXP lamps, so how does the excitation efficiency compare to a HBO lamp in practice? Other advantages, besides long life? Best regards, Jan Grawé ................................................ Jan Grawé Cell Analysis Core Facility Rudbecklaboratoriet/C5 Dag Hammarskjölds väg 20 SE-75185 Uppsala SWEDEN Phone: +(0)18-4714656 Cell:+(0)70-2577874 [hidden email] http://www.rudbeck.uu.se/cellanalys |
 
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Michael Weber-4 |
Re: HXP lamp for fluorescence microscopy
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Jan,
check the information provided by Zeiss: http://zeiss-campus.magnet.fsu.edu/articles/lightsources/metalhalide.html Metal Halide lamps are quite comparable to HBO in terms of output spectra an power, usually a bit more powerful in the blue range. Main advantages to HBO are better stability (less flickering) and longer lifetime, which makes them suitable for life cell imaging. Another option would be LED illumination - less heat output, even longer lifetime, fast switching, more expensive, fixed wavelengths. Michael > Hello, > not a confocal question, but I hope someone may enlighten me. > > One of our users needs to replace the lamp housing on her Zeiss > fluorescence > microscope. The Zeiss rep recommends switching to a HXP lamp and housing. > She needs to know that the HXP lamp will give equal or better excitation > than HBO of especially Alexa 488 and Alexa 546. > I have no experience of HXP lamps, so how does the excitation efficiency > compare to a HBO lamp in practice? Other advantages, besides long life? > > Best regards, > Jan Grawé > > ................................................ > Jan Grawé > Cell Analysis Core Facility > Rudbecklaboratoriet/C5 > Dag Hammarskjölds väg 20 > SE-75185 Uppsala > SWEDEN > Phone: +(0)18-4714656 > Cell:+(0)70-2577874 > [hidden email] > http://www.rudbeck.uu.se/cellanalys |
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Peter Gabriel Pitrone |
Re: HXP lamp for fluorescence microscopy
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In reply to this post by Jan Grawe
Hello Jan,
If this HXO is a metal halide lamp (which I doubt), then the spectral properties are better in the green excitation with shallower UV peaks compared to a normal Hg burner. If it is a Xenon lamp (which I think it is), then it is pretty flat but powerful through out the UV/visible spectrum with the largest spikes in the IR (which are filtered out before it goes through the scope anyway). Pete On Jun 2, 2010, at 09:43 AM, Jan Grawé wrote: > Hello, > not a confocal question, but I hope someone may enlighten me. > > One of our users needs to replace the lamp housing on her Zeiss fluorescence > microscope. The Zeiss rep recommends switching to a HXP lamp and housing. > She needs to know that the HXP lamp will give equal or better excitation > than HBO of especially Alexa 488 and Alexa 546. > I have no experience of HXP lamps, so how does the excitation efficiency > compare to a HBO lamp in practice? Other advantages, besides long life? > > Best regards, > Jan Grawé > > ................................................ > Jan Grawé > Cell Analysis Core Facility > Rudbecklaboratoriet/C5 > Dag Hammarskjölds väg 20 > SE-75185 Uppsala > SWEDEN > Phone: +(0)18-4714656 > Cell:+(0)70-2577874 > [hidden email] > http://www.rudbeck.uu.se/cellanalys |
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Olga Makarova |
DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Olga Makarova
Hi everybody,
I am trying to use DeltaVision environmental chamber 40X, time lapse to figure out localization of my EYFR-tagged protein. I did it once. When I fixed cells , endogenous protein predominantly is in the nucleus and also in cytoplasm, but sometimes only in cytoplasm. EGFP-tagged also shows the same pattern. I thought that transition could depend on stage of cells, particularly mitosis. I am going to include DIC imaging. I also want to add DNA dye for the living cells. What would be the best choice? Or are there any other dyes to track different stages of cells? I am using transiently transfected HPV epithelial cells which divided once in 4 days. Probably I need some synchronization before or after transfection. What would be a better choice. Also I found that media evaporated rather quickly in the chamber making media ingredients more concentrated. Any advice regarding that issue? Thank you very much for any advise. 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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Artem Pliss |
Re: DeltaVision, mitosis, EYFP-tagged protein
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Hi Olga,
You may apply a drop of mineral oil on top of your medium- this would reduce the evaporation. For live cells DNA stain most people use Hoechst 33342, you may also refer to this article: Martin et al (2005) DNA labeling in living cells. Cytometry A 67:45–52. In my experience long term applications of any DNA dye reduces the cell viability and may prevent them from dividing. As an alternative you may consider histone- fluorescent proteins fusion. Best, Art Regarding the DNA dye Hoechst usually works well- but On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova <[hidden email]> wrote: Hi everybody, |
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Olga Makarova |
Re: DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Olga Makarova
Hi all,
This is some additional questions about live cell image. Which live cells dye is better Hoechst 33342 or AMCA? Which one does effect mitosis the least. Why do all cells seems to crawl very quickly to one direction making it practically impossible to monitor during few hours. Thank you, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Olga Makarova <[hidden email]> 8/6/2010 2:10 PM >>> Hi everybody, I am trying to use DeltaVision environmental chamber 40X, time lapse to figure out localization of my EYFR-tagged protein. I did it once. When I fixed cells , endogenous protein predominantly is in the nucleus and also in cytoplasm, but sometimes only in cytoplasm. EGFP-tagged also shows the same pattern. I thought that transition could depend on stage of cells, particularly mitosis. I am going to include DIC imaging. I also want to add DNA dye for the living cells. What would be the best choice? Or are there any other dyes to track different stages of cells? I am using transiently transfected HPV epithelial cells which divided once in 4 days. Probably I need some synchronization before or after transfection. What would be a better choice. Also I found that media evaporated rather quickly in the chamber making media ingredients more concentrated. Any advice regarding that issue? Thank you very much for any advise. 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 ********************************************************** 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 Makarova |
Re: DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Artem Pliss
Thank you , Tom,
How come CO2 will penetrate through mineral oil? How quickly DNA dye effect mitosis? Does histone- fluorescent proteins available commercially? Do you mean to cotransfect histone- fluorescent proteins DNA along with my tagged protein? Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Artem Pliss <[hidden email]> 8/6/2010 5:53 PM >>> Hi Olga, You may apply a drop of mineral oil on top of your medium- this would reduce the evaporation. For live cells DNA stain most people use Hoechst 33342, you may also refer to this article: Martin et al (2005) DNA labeling in living cells. Cytometry A 67:45–52. In my experience long term applications of any DNA dye reduces the cell viability and may prevent them from dividing. As an alternative you may consider histone- fluorescent proteins fusion. Best, Art * *Regarding the DNA dye Hoechst usually works well- but On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova <[hidden email]>wrote: > Hi everybody, > > I am trying to use DeltaVision environmental chamber 40X, time lapse to > figure out localization of my EYFR-tagged protein. I did it once. > When I fixed cells , endogenous protein predominantly is in the nucleus > and also in cytoplasm, but sometimes only in cytoplasm. > EGFP-tagged also shows the same pattern. I thought that transition could > depend on stage of cells, particularly mitosis. > I am going to include DIC imaging. I also want to add DNA dye for the > living cells. What would be the best choice? Or are there any other dyes to > track different stages of cells? > I am using transiently transfected HPV epithelial cells which divided once > in 4 days. Probably I need some synchronization before or after > transfection. What would be a better choice. > Also I found that media evaporated rather quickly in the chamber making > media ingredients more concentrated. Any advice regarding that issue? > Thank you very much for any advise. > > Olga > > > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should > 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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Clements, Ian |
Re: DeltaVision, mitosis, EYFP-tagged protein
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The shortest path to getting a histone or nuclear targeted FP-fusion into your cells would be to use the CellularLights products from invitrogen/Life Technologies. These are BacMan viruses encoding fluorescent proteins tagged with various targeting sequences designed to put the protein into particular cellular compartments or locations. The BacMam baculovirus system is used for delivery, so no engineering or molecular biology required.
Being a virus system some cell types may have different transfection efficiencies but check the Invitrogen website for more details. Basically add the supplied virus suspension to your cell culture and within 24 hrs your cells will be expressing the marker you desire. Ian Clements Product Manager – DeltaVision|OMX® Systems -----Original Message----- From: Olga Makarova [mailto:[hidden email]] Sent: Monday, August 09, 2010 9:54 AM To: [hidden email] Subject: Re: DeltaVision, mitosis, EYFP-tagged protein Thank you , Tom, How come CO2 will penetrate through mineral oil? How quickly DNA dye effect mitosis? Does histone- fluorescent proteins available commercially? Do you mean to cotransfect histone- fluorescent proteins DNA along with my tagged protein? Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Artem Pliss <[hidden email]> 8/6/2010 5:53 PM >>> Hi Olga, You may apply a drop of mineral oil on top of your medium- this would reduce the evaporation. For live cells DNA stain most people use Hoechst 33342, you may also refer to this article: Martin et al (2005) DNA labeling in living cells. Cytometry A 67:45–52. In my experience long term applications of any DNA dye reduces the cell viability and may prevent them from dividing. As an alternative you may consider histone- fluorescent proteins fusion. Best, Art * *Regarding the DNA dye Hoechst usually works well- but On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova <[hidden email]>wrote: > Hi everybody, > > I am trying to use DeltaVision environmental chamber 40X, time lapse to > figure out localization of my EYFR-tagged protein. I did it once. > When I fixed cells , endogenous protein predominantly is in the nucleus > and also in cytoplasm, but sometimes only in cytoplasm. > EGFP-tagged also shows the same pattern. I thought that transition could > depend on stage of cells, particularly mitosis. > I am going to include DIC imaging. I also want to add DNA dye for the > living cells. What would be the best choice? Or are there any other dyes to > track different stages of cells? > I am using transiently transfected HPV epithelial cells which divided once > in 4 days. Probably I need some synchronization before or after > transfection. What would be a better choice. > Also I found that media evaporated rather quickly in the chamber making > media ingredients more concentrated. Any advice regarding that issue? > Thank you very much for any advise. > > Olga > > > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should > 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 This email message, together with any attachments, is for the sole use of the intended recipient(s) and is the confidential information of Applied Precision Inc. If you are not the intended recipient, your review, use, disclosure, copying or dissemination of this email message or its attachments, or the information contained therein, is strictly prohibited. If you are not the intended recipient or if you think this email was sent to you in error, please notify the sender by reply email and delete this message and its attachments, as well as all copies, from your system. |
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Julio Vazquez |
Re: DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Olga Makarova
Hi Olga,
I haven't heard of AMCA per se being used as a nuclear specific dye. As for Hoechst (or DAPI), some cells tolerate it better than others. However, imaging these dyes requires UV, which is pretty toxic, so your cells won't last very long under those conditions... it all depends on how much and how long you need to image them. One alternative could be DRAQ5, which is imaged in the far red. You may want to check the info at this site: and maybe check the paper by Martin et al., CytometryPart A 67A: 45-52 (2005), and also other refs in the biostatus site. For media evaporation, you may want to increase the volume, or use a humidifier inside your incubation chamber. Some microscope incubation systems provide humidity. A bunch of kimwipes dipped into bottles with water or buffer inside the chamber may provide an OK solution for a few hours. You can check the Invitrogen/molecular probes web site, BD biosciences, and other suppliers for cell cycle markers: Without fancy cell cycle indicators, a simple quantitation of nuclear DNA staining may tell you if cells are in G1 or G2, and chromatin condensation may indicate M phase (but possibly also apoptosis). If cells are undergoing mitosis, you will probably see it in your time lapse movies. DIC imaging is a good idea, since it's less toxic for cells. You could use it for much of the imaging, and only use fluorescence fro a subset of your time points. If you are new to live cell imaging, you may want to do some background reading such as "Live Cell Imaging: A laboratory manual" from Cold Spring Harbor laboratory Press, or consult with your University's microscopy facility. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Aug 9, 2010, at 9:48 AM, Olga Makarova wrote:
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Artem Pliss |
Re: DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Olga Makarova
Olga,
1) CO2 does not penetrate through the mineral oil, but in case that cells are already attached, you do not even need it. Given that you have enough cells, they will maintain pH on their own. I was incubating cells (at ~50% confluency) in this system overnight and cells were normally dividing. However, I would use this system only in emergency, I hope that humidification in your incubation system can be fixed. 2) Choice of DNA counterstain depends on your tasks. In case that your observation time is short, just few hours, I see no problem in using the Hoechst. However, if you have to follow the cells for longer intervals, Hoechst as well as any other known to me dye may interfere with the cell cycle progression and with cells viability. In case that you are performing really long imaging sessions you may consider cells transfection with histones tagged to fluorescence proteins. You may find an affordable source of constructs ready for amplification and use at http://www.addgene.org/ Best, Art On Mon, Aug 9, 2010 at 12:53 PM, Olga Makarova <[hidden email]> wrote: Thank you , Tom, |
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James Denegre |
Re: CO2, mineral oil and live-cell imaging
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We routinely use a mineral oil overlay on 100 ul drops of medium to prevent evaporation during long-term (3-4 days) time-lapse imaging of mouse embryos. The CO2 does penetrate the mineral oil, it is a liquid in which the absorption coefficients of CO2 and O2 are similar to those in water (Kubie, JBC, 1927). We equilibrate the dishes for at least an hour in a 5% CO2/5% O2 balance N2 gassed incubator prior to adding embryos, then add embryos and begin imaging. We do not need to exchange medium during the experiment. You might find in necessary to condition the mineral oil with medium by o/n stirring together, to prevent extraction into the mineral oil of components of the medium. We have been using Sigma mineral oil with no need for conditioning. We find DRAQ 5, DAPI and Hoechst to be toxic, don’t know if it is the UV laser, the dye or both. I strongly recommend the idea of using histone fluorescent fusion proteins if you can. Regards, Jim James Denegre, Ph.D. Senior Manager Imaging Sciences The Jackson Laboratory Bar Harbor, ME 04609 207.288.6648 On 8/9/10 1:59 PM, "Artem Pliss" <ampliss@...> wrote: Olga, |
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Clements, Ian |
Re: DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Olga Makarova
Dear Olga,
Re: Why do all cells seems to crawl very quickly to one direction making it practically impossible to monitor during few hours. DeltaVision software has a Cell Tracking Feature designed to answer this problem. The Cell Tracking function will move the stage and follow cells as they move during time-lapse experiments. The Cell Tracking feature is easy to set up and can follow even fast moving cells. Kathryn Buchanan, Ph.D DeltaVision Product Manager Applied Precision, Inc. -----Original Message----- From: Olga Makarova [mailto:[hidden email]] Sent: Monday, August 09, 2010 9:48 AM To: [hidden email] Subject: Re: DeltaVision, mitosis, EYFP-tagged protein Hi all, This is some additional questions about live cell image. Which live cells dye is better Hoechst 33342 or AMCA? Which one does effect mitosis the least. Why do all cells seems to crawl very quickly to one direction making it practically impossible to monitor during few hours. Thank you, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Olga Makarova <[hidden email]> 8/6/2010 2:10 PM >>> Hi everybody, I am trying to use DeltaVision environmental chamber 40X, time lapse to figure out localization of my EYFR-tagged protein. I did it once. When I fixed cells , endogenous protein predominantly is in the nucleus and also in cytoplasm, but sometimes only in cytoplasm. EGFP-tagged also shows the same pattern. I thought that transition could depend on stage of cells, particularly mitosis. I am going to include DIC imaging. I also want to add DNA dye for the living cells. What would be the best choice? Or are there any other dyes to track different stages of cells? I am using transiently transfected HPV epithelial cells which divided once in 4 days. Probably I need some synchronization before or after transfection. What would be a better choice. Also I found that media evaporated rather quickly in the chamber making media ingredients more concentrated. Any advice regarding that issue? Thank you very much for any advise. 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 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues This email message, together with any attachments, is for the sole use of the intended recipient(s) and is the confidential information of Applied Precision Inc. If you are not the intended recipient, your review, use, disclosure, copying or dissemination of this email message or its attachments, or the information contained therein, is strictly prohibited. If you are not the intended recipient or if you think this email was sent to you in error, please notify the sender by reply email and delete this message and its attachments, as well as all copies, from your system. |
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Ignatius, Mike-2 |
Re: DeltaVision, mitosis, EYFP-tagged protein *Vendor Response*
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In reply to this post by Clements, Ian
"Or are there any other dyes to track different stages of cells?"
In the Cellular Lights products generously mentioned below, there is one that fits this specific task - Premo(tm) FUCCI Cell Cycle Sensor Cat. No. P36232. Developed by Miyawaki and colleagues, the fluorescence ubiquitination cell cycle indicator (FUCCI), is a genetically encoded, two-color (red and green), indicator that lets you follow cell division within a cell population. http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-and-Tissue-Analysis/Cell-Viability-and-Regulation/CVPF-Misc/Live-Cell-Imaging-of-Cell-Cycle-and-Division.html Mike Ignatius, Molecular Probes/Lifetech. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Clements, Ian Sent: Monday, August 09, 2010 10:18 AM To: [hidden email] Subject: Re: DeltaVision, mitosis, EYFP-tagged protein The shortest path to getting a histone or nuclear targeted FP-fusion into your cells would be to use the CellularLights products from invitrogen/Life Technologies. These are BacMan viruses encoding fluorescent proteins tagged with various targeting sequences designed to put the protein into particular cellular compartments or locations. The BacMam baculovirus system is used for delivery, so no engineering or molecular biology required. Being a virus system some cell types may have different transfection efficiencies but check the Invitrogen website for more details. Basically add the supplied virus suspension to your cell culture and within 24 hrs your cells will be expressing the marker you desire. Ian Clements Product Manager - DeltaVision|OMX(r) Systems -----Original Message----- From: Olga Makarova [mailto:[hidden email]] Sent: Monday, August 09, 2010 9:54 AM To: [hidden email] Subject: Re: DeltaVision, mitosis, EYFP-tagged protein Thank you , Tom, How come CO2 will penetrate through mineral oil? How quickly DNA dye effect mitosis? Does histone- fluorescent proteins available commercially? Do you mean to cotransfect histone- fluorescent proteins DNA along with my tagged protein? Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Artem Pliss <[hidden email]> 8/6/2010 5:53 PM >>> Hi Olga, You may apply a drop of mineral oil on top of your medium- this would reduce the evaporation. For live cells DNA stain most people use Hoechst 33342, you may also refer to this article: Martin et al (2005) DNA labeling in living cells. Cytometry A 67:45-52. In my experience long term applications of any DNA dye reduces the cell viability and may prevent them from dividing. As an alternative you may consider histone- fluorescent proteins fusion. Best, Art * *Regarding the DNA dye Hoechst usually works well- but On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova <[hidden email]>wrote: > Hi everybody, > > I am trying to use DeltaVision environmental chamber 40X, time lapse to > figure out localization of my EYFR-tagged protein. I did it once. > When I fixed cells , endogenous protein predominantly is in the nucleus > and also in cytoplasm, but sometimes only in cytoplasm. > EGFP-tagged also shows the same pattern. I thought that transition could > depend on stage of cells, particularly mitosis. > I am going to include DIC imaging. I also want to add DNA dye for the > living cells. What would be the best choice? Or are there any other dyes to > track different stages of cells? > I am using transiently transfected HPV epithelial cells which divided once > in 4 days. Probably I need some synchronization before or after > transfection. What would be a better choice. > Also I found that media evaporated rather quickly in the chamber making > media ingredients more concentrated. Any advice regarding that issue? > Thank you very much for any advise. > > Olga > > > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should > 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 This email message, together with any attachments, is for the sole use of the intended recipient(s) and is the confidential information of Applied Precision Inc. If you are not the intended recipient, your review, use, disclosure, copying or dissemination of this email message or its attachments, or the information contained therein, is strictly prohibited. If you are not the intended recipient or if you think this email was sent to you in error, please notify the sender by reply email and delete this message and its attachments, as well as all copies, from your system. |
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Olga Makarova |
Re: DeltaVision, mitosis, EYFP-tagged protein
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In reply to this post by Olga Makarova
Thank you very much to all of you for your comments.
Sorry that I did not reply to some of you. We did not get a grant and have to stop doing live imaging. Thanks, Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Olga Makarova <[hidden email]> 8/9/2010 12:53 PM >>> Thank you , Tom, How come CO2 will penetrate through mineral oil? How quickly DNA dye effect mitosis? Does histone- fluorescent proteins available commercially? Do you mean to cotransfect histone- fluorescent proteins DNA along with my tagged protein? Olga Olga Makarova, PhD Research Specialist 5323 MSRB III >>> Artem Pliss <[hidden email]> 8/6/2010 5:53 PM >>> Hi Olga, You may apply a drop of mineral oil on top of your medium- this would reduce the evaporation. For live cells DNA stain most people use Hoechst 33342, you may also refer to this article: Martin et al (2005) DNA labeling in living cells. Cytometry A 67:45–52. In my experience long term applications of any DNA dye reduces the cell viability and may prevent them from dividing. As an alternative you may consider histone- fluorescent proteins fusion. Best, Art * *Regarding the DNA dye Hoechst usually works well- but On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova <[hidden email]>wrote: > Hi everybody, > > I am trying to use DeltaVision environmental chamber 40X, time lapse to > figure out localization of my EYFR-tagged protein. I did it once. > When I fixed cells , endogenous protein predominantly is in the nucleus > and also in cytoplasm, but sometimes only in cytoplasm. > EGFP-tagged also shows the same pattern. I thought that transition could > depend on stage of cells, particularly mitosis. > I am going to include DIC imaging. I also want to add DNA dye for the > living cells. What would be the best choice? Or are there any other dyes to > track different stages of cells? > I am using transiently transfected HPV epithelial cells which divided once > in 4 days. Probably I need some synchronization before or after > transfection. What would be a better choice. > Also I found that media evaporated rather quickly in the chamber making > media ingredients more concentrated. Any advice regarding that issue? > Thank you very much for any advise. > > Olga > > > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > > ********************************************************** > Electronic Mail is not secure, may not be read every day, and should > 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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Valeria Berno-2 |
Re: CO2, mineral oil and live-cell imaging
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In reply to this post by James Denegre
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** On 09/08/2010 20:16, James Denegre wrote: > Hello Olga, > > We routinely use a mineral oil overlay on 100 ul drops of medium to > prevent evaporation during long-term (3-4 days) time-lapse imaging of > mouse embryos. The CO2 does penetrate the mineral oil, it is a liquid > in which the absorption coefficients of CO2 and O2 are similar to > those in water (Kubie, JBC, 1927). We equilibrate the dishes for at > least an hour in a 5% CO2/5% O2 balance N2 gassed incubator prior to > adding embryos, then add embryos and begin imaging. We do not need to > exchange medium during the experiment. > > You might find in necessary to condition the mineral oil with medium > by o/n stirring together, to prevent extraction into the mineral oil > of components of the medium. We have been using Sigma mineral oil > with no need for conditioning. > > We find DRAQ 5, DAPI and Hoechst to be toxic, don't know if it is the > UV laser, the dye or both. I strongly recommend the idea of using > histone fluorescent fusion proteins if you can. > > Regards, > > Jim > > James Denegre, Ph.D. > Senior Manager > Imaging Sciences > The Jackson Laboratory > Bar Harbor, ME 04609 > 207.288.6648 > > > > > On 8/9/10 1:59 PM, "Artem Pliss" <[hidden email]> wrote: > > Olga, > 1) CO2 does not penetrate through the mineral oil, but in case > that cells are already attached, you do not even need it. Given > that you have enough cells, they will maintain pH on their own. I > was incubating cells (at ~50% confluency) in this system > overnight and cells were normally dividing. However, I would use > this system only in emergency, I hope that humidification in your > incubation system can be fixed. > 2) Choice of DNA counterstain depends on your tasks. In case that > your observation time is short, just few hours, I see no problem > in using the Hoechst. However, if you have to follow the cells for > longer intervals, Hoechst as well as any other known to me dye may > interfere with the cell cycle progression and with cells > viability. In case that you are performing really long imaging > sessions you may consider cells transfection with histones tagged > to fluorescence proteins. > You may find an affordable source of constructs ready for > amplification and use at http://www.addgene.org/ > Best, > Art > > On Mon, Aug 9, 2010 at 12:53 PM, Olga Makarova > <[hidden email]> wrote: > > Thank you , Tom, > > How come CO2 will penetrate through mineral oil? > How quickly DNA dye effect mitosis? > Does histone- fluorescent proteins available commercially? Do > you mean > to cotransfect histone- fluorescent proteins DNA along with my > tagged > protein? > > Olga > > Olga Makarova, PhD > Research Specialist > 5323 MSRB III > > > >>> Artem Pliss <[hidden email]> 8/6/2010 5:53 PM >>> > Hi Olga, > You may apply a drop of mineral oil on top of your medium- > this would > reduce > the evaporation. For live cells DNA stain most people use Hoechst > 33342, you > may also refer to this article: Martin et al (2005) DNA > labeling in > living > cells. Cytometry A 67:45--52. In my experience long term > applications > of any > DNA dye reduces the cell viability and may prevent them from > dividing. > As an > alternative you may consider histone- fluorescent proteins fusion. > Best, > Art > > > > * *Regarding the DNA dye Hoechst usually works well- but > > On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova > <[hidden email]>wrote <[hidden email]%3Ewrote>: > > > Hi everybody, > > > > I am trying to use DeltaVision environmental chamber 40X, > time lapse > to > > figure out localization of my EYFR-tagged protein. I did it > once. > > When I fixed cells , endogenous protein predominantly is in the > nucleus > > and also in cytoplasm, but sometimes only in cytoplasm. > > EGFP-tagged also shows the same pattern. I thought that > transition > could > > depend on stage of cells, particularly mitosis. > > I am going to include DIC imaging. I also want to add DNA dye for > the > > living cells. What would be the best choice? Or are there any > other > dyes to > > track different stages of cells? > > I am using transiently transfected HPV epithelial cells which > divided > once > > in 4 days. Probably I need some synchronization before or after > > transfection. What would be a better choice. > > Also I found that media evaporated rather quickly in the chamber > making > > media ingredients more concentrated. Any advice regarding that > issue? > > Thank you very much for any advise. > > > > 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 > > > ********************************************************** > Electronic Mail is not secure, may not be read every day, and > should not be used for urgent or sensitive issues > > > Valeria Berno,PhD Microscopy Facility Manager MRC Centre for Regenerative Medicine Institute for Stem Cell Research University of Edinburgh Roger Land Building West Mains Road Edinburgh EH9 3JQ Rm 273 Tel Office 0131 6517265 ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. |
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