[Histonet] RE: chromogens for triple immunohistochemistry

[C.M. van der Loos]

Dear Anjan Kumar,IHC triple staining based on three different chromogens is possible with using various combinations of DAB (HRP), Vector VIP (HRP), Liquid Permanent Red (AP), Vector Blue, X-gal (beta-GAL). As long as the primary antibodies of interest do no show co-localization, you may end up with a reasonable staining result of three chromogens that (depending on the choice of chromogens) could be rather well discriminated from each other. However, as you said that all 'antigens of interest show co-localization',  you do have a probem. Most chromogens that mix, do not have a distinct color that could be easy discriminated from both basic colors. The only way to solve this problem is the performance of spectral imaging analysis. Go to the Cambridge Research Instrumentation website for more info (www.cri-inc.com/products/nuance ). Under support/FAQs you will find a PDF (van der Loos pamphlet on multiple staining) that deals with the different chromogens for spectral imaging. Cheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands   Date: Thu, 8 Jan 2009 17:05:52 +0530
From: anjan kumar <drvet_anjan <@t> hotmail.com>
Subject: [Histonet] chromogens for triple immunohistochemistry
To: triple immunohistochem <histonet <@t> lists.utsouthwestern.edu>

hi,
   I am very thankful to everyone for their replies to my questions.
 i had another one which is quite confusing and it is like i wanted to know which three colour chromogen combinations is best for triple immunohistochemistry. 
As all  antigen of my interest are all co localized.
regards,
anjan kumar,
Junior research scientist,
Madras veterinary college.