[Histonet] Stripping antibodies

[C.M. van der Loos]

Amos and John,
I do not agree with John saying that "antibodies could be easily removed by acidity pH1-2". High affinity primaries will stay at the tissue section nicely bound to the epitope. Try for example SMA, clone 1A4 antibody in the usual dilution. After this incubation strip off the antibody by glycin-HCl buffer pH2 and then apply an anti-mouse detection system. You will stain SMA for sure. The only way to get antibodies off is 10 min boiling with citrate pH6.0 or so, as in HIER. I have described that in JOH in 2008, vol 31, pp. 119-127, being the basis of an AP sequential double staining.

Chris van der Loos
Academic Medical Center
Dept. of Pathology M2-230
Amsterdam
The Netherlands

Date: Sun, 30 Oct 2011 12:09:58 -0400
From: John Kiernan <jkiernan <@t> uwo.ca>
Subject: Re: [Histonet] Stripping antibodies
To: histonet <@t> lists.utsouthwestern.edu, cweston <@t> valasciences.com
Cc: Amos Brooks <amosbrooks <@t> gmail.com>
Message-ID: <7640acec48487.4ead3e96 <@t> uwo.ca>
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Antibodies are quite easily removed by acidity - pH 1 to 2. This also removes enzyme-antibody conjugates. The coloured products of the enzyme histochemical reactions are generally not affected. The brown oxidation product of DAB (from peroxidase) and the blue products from bromochloroindoxyl/tetrazolium methods (for alkaline phosphatase) are very stable. This is good: you can remove all the primary and enzyme-labelled antibodies without losing track of the stained antigen in the section. A second immunostaining, with a differently coloured end-product can follow.  If you use AEC as a peroxidase chromogen (red), it should be for the last immunohistochemical procedure, and you will have to use an aqueous mounting medium. The AEC oxidation product dissolves in alcohol and other organic solvents.
 
Amos suggests a strong oxidation (acid permanganate followed by oxalic acid to remove brown manganese dioxide) to get rid of the insoluble brown oxidation product of DAB. Is this what you want?  This oxidation converts the sulphur-containing amino acids, including cystine -S-S- links, to sulphonic acids, which attract cationic dyes even at pH 1. This oxidation couuld seriously modify the epitopes for the next applied primary antibody.
 
Multicolour immunohistochemistry has been a developed technology for several years. Any lab needing to do such work needs to take training courses (hundreds of dollars) or buy a book (tens of dollars).  The one by Chris van der Loos, Immunoenzyme Multiple Staining Methods, Oxford, UK: Bios,  1999, is very good.
 
John Kiernan
Anatomy, UWO
London, Canada