Alexa488 staining question
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Hello All,
This is a question about using Molecualr Probes Alexa 488 Donkey Anti-Mouse secondary antibodies.
I am using this antidody to detect a number of different targets and have noticed that the signal intensity fades over the course of 7-10 days.
I am using it for indirect immunofluorescent detection of targets that form intra-nuclear foci ranging in size from ~300nm to 2um. Compared to the distinct, punctate, bright
foci I image within 1-2 days after staining, 7-10 days later they are smaller, more diffuse and not bright at all. Using the same exposure times and gain setting does not yeild the same quality of images.
This is an obvious problem as I am trying to be consistent with my settings from sample to sample for quantitation.
Has anyone come across a problem like this previously? I am mounting my coverslips with Vectashield. Has anyone come across an antifade mountant that has an RI closer to that of immersion oil?
Thanks to all.
Cheers
Farid
-- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal It sounds like using Prolong Gold from Molecular Probes (Invitrogen) would make the most sense in your situation. If used correctly, it would certainly help with the Alexa 488 signal and provides a RI that should be matched to your immersion oil. No commercial affiliation. -- Samuel A. Connell Director, Light Microscopy Cell & Tissue Imaging Center St. Jude Children's Research Hospital 332 North Lauderdale St., E7061 Memphis, TN 38105-2794 (901) 495-2536 [hidden email] -----Original Message----- From: Confocal Microscopy List on behalf of Farid Jalali Sent: Sat 10/20/2007 10:22 AM To: [hidden email] Subject: Alexa488 staining question. . Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello All, This is a question about using Molecualr Probes Alexa 488 Donkey Anti-Mouse secondary antibodies. I am using this antidody to detect a number of different targets and have noticed that the signal intensity fades over the course of 7-10 days. I am using it for indirect immunofluorescent detection of targets that form intra-nuclear foci ranging in size from ~300nm to 2um. Compared to the distinct, punctate, bright foci I image within 1-2 days after staining, 7-10 days later they are smaller, more diffuse and not bright at all. Using the same exposure times and gain setting does not yeild the same quality of images. This is an obvious problem as I am trying to be consistent with my settings from sample to sample for quantitation. Has anyone come across a problem like this previously? I am mounting my coverslips with Vectashield. Has anyone come across an antifade mountant that has an RI closer to that of immersion oil? Thanks to all. Cheers Farid -- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
 
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Michael Schell |
Re: Alexa488 staining question. .
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I agree. Lose the Vectashield. Switching to Prolong Gold will allow you to preserve your signal for months. None of the mounting medias for fluorescence will completely solve your RI mismatch. Prolong, for example, goes from 1.39 to 1.45 as it dries (see the Invitrogen spec sheet), but you will never reach 1.518 (oil). Mowiol is a bit closer (1.49), and Pawley says that 84% sucrose is the same as oil. Has anybody successfully implemented 84% sucrose as a fluorescent mounting medium? No commercial affiliation. Michael J. Schell, Assist. Professor Dept. of Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814-3220 tel: (301) 295-3249 [hidden email] On Oct 20, 2007, at 12:26 PM, Connell, Samuel wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > It sounds like using Prolong Gold from Molecular Probes > (Invitrogen) would make the most sense in your situation. If used > correctly, it would certainly help with the Alexa 488 signal and > provides a RI that should be matched to your immersion oil. > > No commercial affiliation. > > -- > Samuel A. Connell > Director, Light Microscopy > Cell & Tissue Imaging Center > St. Jude Children's Research Hospital > 332 North Lauderdale St., E7061 > Memphis, TN 38105-2794 > (901) 495-2536 > [hidden email] > > > > > > -----Original Message----- > From: Confocal Microscopy List on behalf of Farid Jalali > Sent: Sat 10/20/2007 10:22 AM > To: [hidden email] > Subject: Alexa488 staining question. . > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello All, > This is a question about using Molecualr Probes Alexa 488 Donkey > Anti-Mouse > secondary antibodies. > I am using this antidody to detect a number of different targets > and have > noticed that the signal intensity fades over the course of 7-10 days. > I am using it for indirect immunofluorescent detection of targets > that form > intra-nuclear foci ranging in size from ~300nm to 2um. Compared to > the distinct, punctate, bright > foci I image within 1-2 days after staining, 7-10 days later they are > smaller, more diffuse and not bright at all. Using the same > exposure times > and gain setting does not yeild the same quality of images. > This is an obvious problem as I am trying to be consistent with my > settings > from sample to sample for quantitation. > > Has anyone come across a problem like this previously? I am > mounting my > coverslips with Vectashield. Has anyone come across an antifade > mountant > that has an RI closer to that of immersion oil? > > Thanks to all. > Cheers > Farid > > -- > Farid Jalali MSc > Senior Research Technician/ Lab Manager > Dr. Robert Bristow Lab > Applied Molecular Oncology > Princess Margaret Hospital > Toronto, Canada > 416-946-4501 X4351 (Princess Margaret Hospital) > 416-581-7754 STTARR at MaRS Building > 416-581-7791 STTARR Microscopy Suite |
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George McNamara |
Re: Alexa488 staining question
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In reply to this post by Farid Jalali
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Farid, > Has anyone come across an antifade mountant that has an RI closer to that of immersion oil? Thiodiethanol can be adjusted to exactly match RI of immersion oil. See T. Staudt et al Microsc Res Tech 2007. At 11:22 AM 10/20/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >Hello All, >This is a question about using Molecualr Probes Alexa 488 Donkey >Anti-Mouse secondary antibodies. >I am using this antidody to detect a number of different targets and >have noticed that the signal intensity fades over the course of 7-10 days. >I am using it for indirect immunofluorescent detection of targets >that form intra-nuclear foci ranging in size from ~300nm to 2um. >Compared to the distinct, punctate, bright >foci I image within 1-2 days after staining, 7-10 days later they >are smaller, more diffuse and not bright at all. Using the same >exposure times and gain setting does not yeild the same quality of images. >This is an obvious problem as I am trying to be consistent with my >settings from sample to sample for quantitation. > >Has anyone come across a problem like this previously? I am mounting >my coverslips with Vectashield. Has anyone come across an antifade >mountant that has an RI closer to that of immersion oil? > >Thanks to all. >Cheers >Farid > >-- >Farid Jalali MSc >Senior Research Technician/ Lab Manager >Dr. Robert Bristow Lab >Applied Molecular Oncology >Princess Margaret Hospital >Toronto, Canada >416-946-4501 X4351 (Princess Margaret Hospital) >416-581-7754 STTARR at MaRS Building >416-581-7791 STTARR Microscopy Suite George McNamara, Ph.D. University of Miami, Miller School of Medicine Image Core Miami, FL 33010 [hidden email] [hidden email] 305-243-8436 office http://home.earthlink.net/~pubspectra/ http://home.earthlink.net/~geomcnamara/ http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility) |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Thanks alot for this George. This seems really exciting and definitely worthy of a shot.
Thanks to the whole group for their replies.
Farid
On 10/20/07, George McNamara <[hidden email]> wrote:
Search the CONFOCAL archive at -- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Micrroscopy Suite |
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Martin Wessendorf |
Re: Alexa488 staining question
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In reply to this post by Farid Jalali
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Farid-- Farid Jalali wrote: > This is a question about using Molecualr Probes Alexa 488 Donkey > Anti-Mouse secondary antibodies. > I am using this antidody to detect a number of different targets and > have noticed that the signal intensity fades over the course of 7-10 days. > I am using it for indirect immunofluorescent detection of targets that > form intra-nuclear foci ranging in size from ~300nm to 2um. Compared to > the distinct, punctate, bright > foci I image within 1-2 days after staining, 7-10 days later they are > smaller, more diffuse and not bright at all. Using the same exposure > times and gain setting does not yeild the same quality of images. > This is an obvious problem as I am trying to be consistent with my > settings from sample to sample for quantitation. > > Has anyone come across a problem like this previously? I am mounting my > coverslips with Vectashield. Has anyone come across an antifade mountant > that has an RI closer to that of immersion oil? Not sure about the cause of the fading/diffusion of label, but you might try antibodies labeled with Cy2- (or if you can use a red fluorophore, Cy3-) from Jackson ImmunoResearch. You can mount either Cy2, Cy3 or Cy5 using DPX (Fluka) which has an index of refraction reasonably close to that of oil and glass. Martin -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 E-mail: martinw[at]med.umn.edu |
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Martin Wessendorf |
Re: Alexa488 staining question. .
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In reply to this post by Michael Schell
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Michael-- Michael Schell wrote: > (1.49), and Pawley says that 84% sucrose is the same as oil. Has > anybody successfully implemented 84% sucrose as a fluorescent mounting > medium? Yup. We used it for mounting of 300 um thick slices in which cells had been recorded and filled. Sucrose worked fine except for two problems: 1) As it dries down, the coverslip comes closer and closer to the slide. If you're trying to mount relatively thick sections such as we did, the sections will initially look fine. However, after a month or so, the coverslip got sucked down onto the slide with sufficient force that the slice was smashed like a bug on a windshield. This was NOT pretty. However, my guess is that for thinner sections (e.g. 10 um cryostat sections) this wouldn't be a problem. 2) The thick slices initially looked fine and were well-cleared. However, after a month, the tissue was no longer clear--rather, it was cloudy-going-on-opaque. --My guess is that the cause for this had something to do with the increase in sucrose concentration as the water evaporated from the sucrose solution. However, we changed approaches and never pursued this approach further. Good luck! Martin -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 E-mail: martinw[at]med.umn.edu |
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Jacqueline Ross |
Re: Alexa488 staining question and Prolong Gold
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In reply to this post by Michael Schell
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All, Just a quick question about Prolong Gold (with DAPI) and Alexa488. I know Prolong Gold is meant to be very good but I was helping a couple of researchers today imaging their cells, (on coverslips mounted on slides), which had an Alexa488 secondary and they seemed to be bleaching rather rapidly even though they were mounted in Prolong Gold. The samples were actually a couple of weeks old but I have had this experience once before. There also seemed to be a bit of a background problem (in the green channel) aside from any reflection which I was thinking might be something to do with having the DAPI in the mounting medium. The red fluorophore was fine. I thought the Alexa488 bleaching might be caused by having too much residual buffer left on the coverslip prior to mounting which then dilutes down the Prolong Gold? Has anyone else had this experience? Kind regards, Jacqui Jacqueline Ross Biomedical Imaging Research Unit School of Medical Sciences Faculty of Medical & Health Sciences The University of Auckland Private Bag 92019 Auckland, NEW ZEALAND Tel: 64 9 373 7599 Ext 87438 Fax: 64 9 373 7484 http://www.health.auckland.ac.nz/biru/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Schell Sent: 21 October 2007 05:51 To: [hidden email] Subject: Re: Alexa488 staining question. . Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I agree. Lose the Vectashield. Switching to Prolong Gold will allow you to preserve your signal for months. None of the mounting medias for fluorescence will completely solve your RI mismatch. Prolong, for example, goes from 1.39 to 1.45 as it dries (see the Invitrogen spec sheet), but you will never reach 1.518 (oil). Mowiol is a bit closer (1.49), and Pawley says that 84% sucrose is the same as oil. Has anybody successfully implemented 84% sucrose as a fluorescent mounting medium? No commercial affiliation. Michael J. Schell, Assist. Professor Dept. of Pharmacology Uniformed Services University 4301 Jones Bridge Rd. Bethesda, MD 20814-3220 tel: (301) 295-3249 [hidden email] On Oct 20, 2007, at 12:26 PM, Connell, Samuel wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > It sounds like using Prolong Gold from Molecular Probes > (Invitrogen) would make the most sense in your situation. If used > correctly, it would certainly help with the Alexa 488 signal and > provides a RI that should be matched to your immersion oil. > > No commercial affiliation. > > -- > Samuel A. Connell > Director, Light Microscopy > Cell & Tissue Imaging Center > St. Jude Children's Research Hospital > 332 North Lauderdale St., E7061 > Memphis, TN 38105-2794 > (901) 495-2536 > [hidden email] > > > > > > -----Original Message----- > From: Confocal Microscopy List on behalf of Farid Jalali > Sent: Sat 10/20/2007 10:22 AM > To: [hidden email] > Subject: Alexa488 staining question. . > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello All, > This is a question about using Molecualr Probes Alexa 488 Donkey > Anti-Mouse > secondary antibodies. > I am using this antidody to detect a number of different targets > and have > noticed that the signal intensity fades over the course of 7-10 days. > I am using it for indirect immunofluorescent detection of targets > that form > intra-nuclear foci ranging in size from ~300nm to 2um. Compared to > the distinct, punctate, bright > foci I image within 1-2 days after staining, 7-10 days later they are > smaller, more diffuse and not bright at all. Using the same > exposure times > and gain setting does not yeild the same quality of images. > This is an obvious problem as I am trying to be consistent with my > settings > from sample to sample for quantitation. > > Has anyone come across a problem like this previously? I am > mounting my > coverslips with Vectashield. Has anyone come across an antifade > mountant > that has an RI closer to that of immersion oil? > > Thanks to all. > Cheers > Farid > > -- > Farid Jalali MSc > Senior Research Technician/ Lab Manager > Dr. Robert Bristow Lab > Applied Molecular Oncology > Princess Margaret Hospital > Toronto, Canada > 416-946-4501 X4351 (Princess Margaret Hospital) > 416-581-7754 STTARR at MaRS Building > 416-581-7791 STTARR Microscopy Suite |
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Sarah Aubut |
Re: Alexa488 staining question and Prolong Gold
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I recently attended a short course on Basic confocal at the Microscopy meeting in Fort Lauderdale, and the instructors advised against using Prolong and all other mountants that contain polyvinyl alcohol as this causes shrinkage up to 50% in your sample. What they suggested was something that matched the RI of the sample. One specific was PBS + glycerol (30%) with 0.25% p-phenylenediamine. DABCO was also mentioned as an alternative. I don't know if this information helps at all, but I thought I should contribute the little that I know! Sarah Aubut Lab Technician, Microscopy Suite Canadian Forestry Service Sault Ste Marie, ON Canada [hidden email] |
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Judy Trogadis |
Re: Alexa488 staining question and Prolong Gold
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Is sample shrinkage a general problem with mounting media which harden? I am not a fan of the PBS+glycerol+antifade combo because it's so messy - difficult to remove immersion oil, broken bits of nailpolish all over the stage and the lenses . . . . . Judy Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 [hidden email] >>> Sarah Aubut <[hidden email]> 10/23/07 8:56 AM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I recently attended a short course on Basic confocal at the Microscopy meeting in Fort Lauderdale, and the instructors advised against using Prolong and all other mountants that contain polyvinyl alcohol as this causes shrinkage up to 50% in your sample. What they suggested was something that matched the RI of the sample. One specific was PBS + glycerol (30%) with 0.25% p-phenylenediamine. DABCO was also mentioned as an alternative. I don't know if this information helps at all, but I thought I should contribute the little that I know! Sarah Aubut Lab Technician, Microscopy Suite Canadian Forestry Service Sault Ste Marie, ON Canada [hidden email] |
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In reply to this post by Jacqueline Ross
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear All, Prolong Gold is sensitive to humidity in the lab. Backgound levels could vary up to 3-fold (in CFP or FRET channels if samples are not dry, or humidity in the lab is unpredictable). Cover glass should be dry before mounting, and carefully sealed with the nail polish 24 hours post mounting. I image samples 36-48 hours postmounting. SlowFade is a very good alternative, with reproducible background levels, especially if you are prepared to image your samples the same day. Except for the increase of the RI during maturation (1.43-1.48), ProlongGold is a very good media, at least clearly better than Alexa dyes (due to an unknown reason many are unaware of ATTO dyes). Cheers, Vitaly NCI-Frederick ----- Original Message ----- From: "Jacqui Ross" <[hidden email]> To: <[hidden email]> Sent: Monday, October 22, 2007 11:30 PM Subject: Re: Alexa488 staining question and Prolong Gold > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear All, > > Just a quick question about Prolong Gold (with DAPI) and Alexa488. > > I know Prolong Gold is meant to be very good but I was helping a couple > of researchers today imaging their cells, (on coverslips mounted on > slides), which had an Alexa488 secondary and they seemed to be bleaching > rather rapidly even though they were mounted in Prolong Gold. > > The samples were actually a couple of weeks old but I have had this > experience once before. There also seemed to be a bit of a background > problem (in the green channel) aside from any reflection which I was > thinking might be something to do with having the DAPI in the mounting > medium. The red fluorophore was fine. > > I thought the Alexa488 bleaching might be caused by having too much > residual buffer left on the coverslip prior to mounting which then > dilutes down the Prolong Gold? > > Has anyone else had this experience? > > Kind regards, > > Jacqui > > Jacqueline Ross > Biomedical Imaging Research Unit > School of Medical Sciences > Faculty of Medical & Health Sciences > The University of Auckland > Private Bag 92019 > Auckland, NEW ZEALAND > > Tel: 64 9 373 7599 Ext 87438 > Fax: 64 9 373 7484 > > http://www.health.auckland.ac.nz/biru/ > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Michael Schell > Sent: 21 October 2007 05:51 > To: [hidden email] > Subject: Re: Alexa488 staining question. . > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I agree. Lose the Vectashield. Switching to Prolong Gold will allow > you to preserve your signal for months. None of the mounting medias > for fluorescence will completely solve your RI mismatch. Prolong, > for example, goes from 1.39 to 1.45 as it dries (see the Invitrogen > spec sheet), but you will never reach 1.518 (oil). Mowiol is a bit > closer (1.49), and Pawley says that 84% sucrose is the same as oil. > Has anybody successfully implemented 84% sucrose as a fluorescent > mounting medium? > > No commercial affiliation. > > Michael J. Schell, > Assist. Professor > Dept. of Pharmacology > Uniformed Services University > 4301 Jones Bridge Rd. > Bethesda, MD 20814-3220 > tel: (301) 295-3249 > [hidden email] > > On Oct 20, 2007, at 12:26 PM, Connell, Samuel wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> It sounds like using Prolong Gold from Molecular Probes >> (Invitrogen) would make the most sense in your situation. If used >> correctly, it would certainly help with the Alexa 488 signal and >> provides a RI that should be matched to your immersion oil. >> >> No commercial affiliation. >> >> -- >> Samuel A. Connell >> Director, Light Microscopy >> Cell & Tissue Imaging Center >> St. Jude Children's Research Hospital >> 332 North Lauderdale St., E7061 >> Memphis, TN 38105-2794 >> (901) 495-2536 >> [hidden email] >> >> >> >> >> >> -----Original Message----- >> From: Confocal Microscopy List on behalf of Farid Jalali >> Sent: Sat 10/20/2007 10:22 AM >> To: [hidden email] >> Subject: Alexa488 staining question. . >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Hello All, >> This is a question about using Molecualr Probes Alexa 488 Donkey >> Anti-Mouse >> secondary antibodies. >> I am using this antidody to detect a number of different targets >> and have >> noticed that the signal intensity fades over the course of 7-10 days. >> I am using it for indirect immunofluorescent detection of targets >> that form >> intra-nuclear foci ranging in size from ~300nm to 2um. Compared to >> the distinct, punctate, bright >> foci I image within 1-2 days after staining, 7-10 days later they are >> smaller, more diffuse and not bright at all. Using the same >> exposure times >> and gain setting does not yeild the same quality of images. >> This is an obvious problem as I am trying to be consistent with my >> settings >> from sample to sample for quantitation. >> >> Has anyone come across a problem like this previously? I am >> mounting my >> coverslips with Vectashield. Has anyone come across an antifade >> mountant >> that has an RI closer to that of immersion oil? >> >> Thanks to all. >> Cheers >> Farid >> >> -- >> Farid Jalali MSc >> Senior Research Technician/ Lab Manager >> Dr. Robert Bristow Lab >> Applied Molecular Oncology >> Princess Margaret Hospital >> Toronto, Canada >> 416-946-4501 X4351 (Princess Margaret Hospital) >> 416-581-7754 STTARR at MaRS Building >> 416-581-7791 STTARR Microscopy Suite > |
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Rosemary.White |
Re: Alexa488 staining question and Prolong Gold
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In reply to this post by Judy Trogadis
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, One thing we've tried is Cygel from (forgotten). Melts when cold, solidifies when warm. Only drawback for us, apart from the huge cost, is that it's only sold made up in PBS, rather than the pure compound. May be worth considering as a more permanent mountant, if the coverslip is sealed after mounting. cheers, Rosmeayr Rosemary White [hidden email] CSIRO Plant Industry ph. 61 (0)2-6246 5475 GPO Box 1600 fax. 61 (0)2-6246 5334 Canberra, ACT 2601 Australia On 24/10/07 12:47 AM, "Judy Trogadis" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Is sample shrinkage a general problem with mounting media which harden? I am > not a fan of the PBS+glycerol+antifade combo because it's so messy - difficult > to remove immersion oil, broken bits of nailpolish all over the stage and the > lenses . . . . . > > Judy > > Judy Trogadis > Bio-Imaging Coordinator > St. Michael's Hospital, 7Queen > 30 Bond St. > Toronto, ON M5B 1W8, Canada > ph: 416-864-6060 x6337 > pager: 416-685-9219 > fax: 416-864-6043 > [hidden email] > > >>>> Sarah Aubut <[hidden email]> 10/23/07 8:56 AM >>> > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I recently attended a short course on Basic confocal at the Microscopy > meeting in Fort Lauderdale, and the instructors advised against using > Prolong and all other mountants that contain polyvinyl alcohol as this > causes shrinkage up to 50% in your sample. > What they suggested was something that matched the RI of the sample. One > specific was PBS + glycerol (30%) with 0.25% p-phenylenediamine. DABCO was > also mentioned as an alternative. > I don't know if this information helps at all, but I thought I should > contribute the little that I know! > > > Sarah Aubut > Lab Technician, Microscopy Suite > Canadian Forestry Service > Sault Ste Marie, ON > Canada > > [hidden email] |
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Asson-Batres, Mary Ann |
Re: Alexa488 staining question and Prolong Gold
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In reply to this post by Judy Trogadis
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
We have been using Prolong Anti-Fade for several years and have not had problems with tissue shrinkage using either rat or mouse paraffin-embedded or cyroprotected sections. We have been unhappy with
Prolong Gold because the DAPI inconsistently stains nuclei - sometimes brightly, sometimes not at all, for inexplicable reasons. Thus, we have continued to use the standard Prolong preparation. We provide a separate DAPI treatment when we wish to visualize
nuclei. In our hands, there is little bleaching of FITC or Cyanine dyes and we have been able to retain our stained sections at -20C and image them over and over again for over 2 years using both epifluorescence and confocal laser microscopy at highest intensity.
When we observe background staining on sections, we attribute it to other aspects of the tissue fixation and/or staining processes - not to the prolong antifade. We do not use nail polish or other sealant to seal the coverslip and we observe no drying of
the specimen or changes in image quality after long term storage. The process is clean and the images are clear. It is important to (1) centrifuge the reagent for about 30 min or more before use to ensure it is mixed properly, (2) make the reagent fresh
each day and discard any remaining media, and (3) to apply a very thin coating. It is somewhat costly in terms of money, but in our opinion, worth it, since we can spend a lot of time examining the results without fear of bleaching and we can archive our
results for later reference.
Mary Ann
Mary Ann Asson-Batres
Associate Professor, Biological Sciences Tennessee State University
5300 John A Merritt Blvd
Nashville, TN 37209
ph: 615-963-5779 fax: 615-963-2142
[hidden email] From: Confocal Microscopy List on behalf of Judy Trogadis Sent: Tue 10/23/2007 9:47 AM To: [hidden email] Subject: Re: Alexa488 staining question and Prolong Gold Search the CONFOCAL archive at |
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Ignatius, Mike |
Re: Alexa488 staining question **Vevdor comment**
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In reply to this post by Martin Wessendorf
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Farid, Sorry to be so late in responding, lots of traveling. We tossed your question around here, and one other possibility is that you are seeing some disassociation of the dye from the sample. Antibodies, unless fixed in place, can diffuse away in viscous mountants after a week. You can see this sometimes as diffuse haze over the prep. AF 488, regardless of the conjugate should indeed be as bright on days 1,2 as 7-10. You described, smaller, more diffuse foci, so maybe this is the issue? To avoid this, mountants that harden are preferred. You therefore might want to try ProLong Gold for this application (or try Vectors HardSet mountant.) Prolong Gold cures or hardens to near oil RI values (1.46 after a week). Not 1.5 like oil, but fairly close. The link for more info on Prolong Gold's RI changes during curing is: http://probes.invitrogen.com/media/pis/mp36930.pdf Our SlowFade Gold line remain viscous on the sample, like Vectashield and most home brew formulations. These can be optimal for thick samples, where the RI is mostly determined by the water in the tissue section (1.3). But not thin samples viewed with oil, where 1.5 is sought. Mike Ignatius, Ph.D. Molecular Probes/Invitrogen For additional tech assist question 800 955-6288. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf Sent: Monday, October 22, 2007 6:24 AM To: [hidden email] Subject: Re: Alexa488 staining question Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Farid-- Farid Jalali wrote: > This is a question about using Molecualr Probes Alexa 488 Donkey > Anti-Mouse secondary antibodies. > I am using this antidody to detect a number of different targets and > have noticed that the signal intensity fades over the course of 7-10 days. > I am using it for indirect immunofluorescent detection of targets that > form intra-nuclear foci ranging in size from ~300nm to 2um. Compared to > the distinct, punctate, bright > foci I image within 1-2 days after staining, 7-10 days later they are > smaller, more diffuse and not bright at all. Using the same exposure > times and gain setting does not yeild the same quality of images. > This is an obvious problem as I am trying to be consistent with my > settings from sample to sample for quantitation. > > Has anyone come across a problem like this previously? I am mounting my > coverslips with Vectashield. Has anyone come across an antifade mountant > that has an RI closer to that of immersion oil? Not sure about the cause of the fading/diffusion of label, but you might try antibodies labeled with Cy2- (or if you can use a red fluorophore, Cy3-) from Jackson ImmunoResearch. You can mount either Cy2, Cy3 or Cy5 using DPX (Fluka) which has an index of refraction reasonably close to that of oil and glass. Martin -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 E-mail: martinw[at]med.umn.edu |
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