Re; endogenous peroxidase blocking

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From:bbracing <>
To:Histonet <>
Date:Sat, 27 Mar 1999 15:58:09 -0800 (PST)
Content-Type:text/plain; charset="ISO-8859-1"; X-MAPIextension=".TXT"

Back in the late 80's early 90's when IHC was still in its infancy and about the only reliable IHC method used the P.A.P. technology, I did quite an extensive comparison study on a large number of tumors using many different antibodies, and in many cases it was quite easy to demonstrate that blocking with H2O2 before the primary antibody was applied,  had quite a negative affect on many tumors.  I was also able to demonstrate that H2O2 blocking after the primary antibody, but before the linking antibody also had quite an negative affect on the detection of many tumor antigens. I was not able to demonstrate any negative effects if the blocking step was placed between the link and lable reagents, and so for the past ten or so years this is where we do our endogenous peroxidase blocking.  With the advent of the streptavidin biotin technology, I found that with the increased sensitivity this problem was slightly reduced, but I still kept the blocking step between the link and lable steps. For the last couple of years I have been using the Labelled Polymer (Dako) technology (which is a one step technology that solved several problems we were having with the link lable methods) but I still found it beneficial to block after the primary, but before the labelled polymer.  However, I have not done any comparison studies on tumors which have had heat induced antigen retrieval techiques applied  to them, to see if the placement of the H2O2 blocking step, has any effects on the heat retrieved antigens.  This might be an interesting project?
Anyway, one other point.  I have never been given a satifactory answere as to why the 30% H2O2 in many labs is diluted with absolute, or in some cases 70% alcohol. The naturally occurring  peroxidase enzyme works in an aqueous environment so why not dilute the H2O2 with distilled water, or saline?  I use the former, and to the best of my knowledge it works just fine and is a lot less hassle, as a 25 ml bottle lasts a week, before I have to make up fresh.
Kerry Beebe
Kelowna Gen Hospital
B.C. Canada


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