IHC-rescue ??

From:Heike Grabsch <h.grabsch@uni-koeln.de>

I need some help to "rescue" a immunohistochemical staining I have done 
two days ago. I have been very unlucky this time staining a series of 80 
slides with an monoclonal antibody against ATM. I did pretreatment of the 
slides in the microwave and usually I get a brillinat staining. However, 
this time it was very important and very urgent, so - of course - I ran 
into problems. The first trial was overstained, I used DAB as chromogen. 
Anyhow I counterstained the slides, dehydration and coverslipping 
followed. Then, the second time I used AEC and in about 50 cases very 
very weak staining, so you cannot decide "is this nucleus really 
positive?". My initial thought: destaining by using alcohol to get rid of 
all AEC precipitates completely. This was no problem. Then I did the 
total immunohistochemistry procedure again, even a comparision with and 
without pretreatment. However, the slides were absolutely blank, not even 
a background signal, a positive control ("new slide") was okay. 

Any suggestions? Is the idea in general wrong? I thought I read somewhere 
that you can do several immunohistochemical stainings on the same slide. 

Any help and hint is highly appreciated,

Dr. Heike Grabsch
Dept of Pathology
University of Duesseldorf
Moorenstrasse 5
40225 Duesseldorf

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