|From:||Heike Grabsch <firstname.lastname@example.org>|
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
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