Re: [Histonet] IHC slides losing tissue

From:Rene J Buesa

Sharon:
  If you just air dried the sections on the slides before doing the antigen retrieval in a pressure cooker (where the temperature is >100C) the sections were doomed to fall.
  Just to be clear you don't deparafinize in the oven, what you do in te oven is to melt the paraffin and asure that the sections get in close contact with the glass that, is (+) charged will retain the sections during the IHC procedure.
  To succeed in this step, you have to first make sure that there is no water left between the section and the slide. Thenyou will have to put your slides in a oven that could be at 60C and leave them there for at least hour. Take them out and let them cool to room temperature. After that you can either store them or use them for any staining procedure.
  Sections as thin as the ones you describe (5m) should survive.
  You don't really want to ad gelatin neither on the slide or in the water bath. In
  both instances you will end with background because you have to remember that gelatins are proteins. As a matter of fact the water bath for IHC section should be filled with distilled water alone.  

"Osborn, Sharon"  wrote:
  Histonetters, 
One of our techs cut ovary at 5u paraffin sections placed on charged
slides. These were air dried but not deparaffinized in the oven. The
researcher deparaffinized through zylene then placed in the pressure cooker
at 60 for antigen retrieval. The tissue came off. Our first remedy is to
oven deparaffinize the slides then do the retrieval process. If that is not
successful, what is the solution(s) for this problem. Our senior IHC tech
suggested gelatin subbing on the slides but did not think that would solve
the problem. Would there be too much background with the gelatin should it
be successful? What is your experience in what works best.
Much thanks...
Sharon Osborn
DNAX, SP BioPharma
Palo Alto, CA
650.946.6539


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