Double TSA?
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So I have recently been working on a double staining IHC using TSA to
amplify the signal in one antibody. It's been working great except that as s result of all the washing steps from the TSA protocol the other non TSA antibody stain is too weak now. I have tried to decrease the washing steps which hadn't helped that much. Any suggestions on how I can apply TSA/ amplification to two antibodies? I have Looked around online and haven't found anything concrete. Any suggestions folks have is muchly appreciated. This is using parafin embedded adipose tissue. Thanks! Richard Green |
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It could be an affinity problem with the first anti-body. One
trick to solve this is to re-fix the sample after you have done your first primary anti-body incubation and washes to cross link it to its antigen. Joe Goodhouse Confocal Core Lab Manager Dept. of Molecular Biology Princeton University Washington Road Princeton, NJ. 08544-1014 609-258-5432 Visit us at http://www.molbio1.princeton.edu/facility/confocal/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard Green Sent: Tuesday, December 01, 2009 3:28 PM To: [hidden email] Subject: Double TSA? So I have recently been working on a double staining IHC using TSA to amplify the signal in one antibody. It's been working great except that as s result of all the washing steps from the TSA protocol the other non TSA antibody stain is too weak now. I have tried to decrease the washing steps which hadn't helped that much. Any suggestions on how I can apply TSA/ amplification to two antibodies? I have Looked around online and haven't found anything concrete. Any suggestions folks have is muchly appreciated. This is using parafin embedded adipose tissue. Thanks! Richard Green |
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Hi Richard,
There are two variations of TSA, HRP and AP. I would think if you use HRP for one antibody and AP for the other you should be able to double label. Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Goodhouse, Joseph G. Sent: Wednesday, 2 December 2009 7:36 AM To: [hidden email] Subject: Re: Double TSA? It could be an affinity problem with the first anti-body. One trick to solve this is to re-fix the sample after you have done your first primary anti-body incubation and washes to cross link it to its antigen. Joe Goodhouse Confocal Core Lab Manager Dept. of Molecular Biology Princeton University Washington Road Princeton, NJ. 08544-1014 609-258-5432 Visit us at http://www.molbio1.princeton.edu/facility/confocal/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard Green Sent: Tuesday, December 01, 2009 3:28 PM To: [hidden email] Subject: Double TSA? So I have recently been working on a double staining IHC using TSA to amplify the signal in one antibody. It's been working great except that as s result of all the washing steps from the TSA protocol the other non TSA antibody stain is too weak now. I have tried to decrease the washing steps which hadn't helped that much. Any suggestions on how I can apply TSA/ amplification to two antibodies? I have Looked around online and haven't found anything concrete. Any suggestions folks have is muchly appreciated. This is using parafin embedded adipose tissue. Thanks! Richard Green No virus found in this incoming message. Checked by AVG - www.avg.com Version: 9.0.709 / Virus Database: 270.14.88/2537 - Release Date: 12/02/09 06:32:00 This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd. |
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In reply to this post by Richard Green-2
We published a method for double labeling with TSA and continue to use it with success for cultured cells and different tissues fixed with paraformaldehyde and embedded in paraffin or OCT-frozen sections. You can find it in this reference:
J Comp Neurol. 2006 May 10;496(2):149-71. Localization of retinaldehyde dehydrogenases and retinoid binding proteins to sustentacular cells, glia, Bowman's gland cells, and stroma: potential sites of retinoic acid synthesis in the postnatal rat olfactory organ. at http://www.ncbi.nlm.nih.gov/pubmed/16538685?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=2 Mary Ann Mary Ann Asson-Batres, PhD Professor Department of Biological Sciences Tennessee State University PO Box 1116 (postal mail) Room 313 Harned Hall (courier) 3500 John A Merritt Blvd Nashville, TN 37209 Office 615-963-5779 FAX 615-963-2142 [hidden email] [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Richard Green Sent: Tuesday, December 01, 2009 2:28 PM To: [hidden email] Subject: Double TSA? So I have recently been working on a double staining IHC using TSA to amplify the signal in one antibody. It's been working great except that as s result of all the washing steps from the TSA protocol the other non TSA antibody stain is too weak now. I have tried to decrease the washing steps which hadn't helped that much. Any suggestions on how I can apply TSA/ amplification to two antibodies? I have Looked around online and haven't found anything concrete. Any suggestions folks have is muchly appreciated. This is using parafin embedded adipose tissue. Thanks! Richard Green |
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In reply to this post by Richard Green-2
I haven't tried this protocol but have a reference from
Toth & Mezey in 2007; they were trying to do double-labeling with primaries from the same species & ended up detecting with TSA: J. Histochem. & Cytochem. (55) 6: 545-554 Tamara On Tue, 1 Dec 2009 12:28:14 -0800 Richard Green <[hidden email]> wrote: > So I have recently been working on a double staining IHC >using TSA to amplify the signal in one antibody. It's >been working great except that as s result of all the >washing steps from the TSA protocol the other non TSA >antibody stain is too weak now. I have tried to decrease > the washing steps which hadn't helped that much. Any >suggestions on how I can apply TSA/ amplification to two >antibodies? I have > Looked around online and haven't found anything >concrete. Any suggestions folks have is muchly >appreciated. This is using parafin embedded adipose >tissue. Thanks! > Richard Green *************************** Tamara Howard Cell Biology & Physiology UNM-HSC Albuquerque, NM *************************** |
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Hello..I had the same problem some years ago, so we did a double
amplification using first a biotinilated-FITC marker amplified by an
anti-FITC-ALEXA 433 antibody, then we did the TSA with TRITC as
fluorochrome.
If you wish the method is described here and you may see the photos of the double staining <a href="http://dx.doi.org/10.1006/excr.2001.5342" target="doilink" onclick="var doiWin; doiWin=window.open('http://dx.doi.org/10.1006/excr.2001.5342','doilink','scrollbars=yes,resizable=yes,directories=yes,toolbar=yes,menubar=yes,status=yes'); doiWin.focus()">doi:10.1006/excr.2001.5342 Experimental Cell Research Volume 270, Issue 2, 1 November 2001, Pages 235-247 Good luck Prof. Dr. Francisco Javier Hernandez Blazquez University of São Paulo School of Veterinary Medicine Department of Surgery Av. Prof. Dr. Orlando Marques de Paiva, 87 05508-270 - São Paulo (SP) - Brasil http://www.fmvz.usp.br/index.php/site/docentes/lista_de_docentes/francisco_javier_hernandez_blazquez Tel..55 (11) 3091 1374 Fax 55 (11) 3091 7805 email: [hidden email] Tamara A Howard escreveu: I haven't tried this protocol but have a reference from Toth & Mezey in 2007; they were trying to do double-labeling with primaries from -- |
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