[Histonet] IHC on fresh frozen

Patti Loykasek ploykasek <@t> phenopath.com
Wed Dec 3 10:38:07 CST 2008

After hearing a presentation by Sharon Lear describing some low temp antigen
retrieval that she did, we changed our method for frozen sections. We fix
the frozen sections in 10% nuetral buffered formalin for 30'-60', rinse,
then do a gentle pretreatment. The gentle pretreatment is usually 10mM
citrate buffer pH6 at 70 degrees C for 30'. Slides are cooled, and usual IHC
done. This has worked well for us. Our staining with this method is more
reliable & intense than with previous methods.

Patti Loykasek

> Hi, all
> Recently I did some IHC(chromagen methods) on mouse fresh frozen
> tissues, mainly using insulin antibody on pancreas. The image is
> much fuzzier compare to paraffin embedding tissue. And the
> staining also smeared to acinar cells which surround the islet.
> I airdried slide(>30min) and used a general acetone method(-20C
> 5min) to fix the tissue before I did IHC.
> How can I get a relatively sharp staining on the fresh frozen
> tissue?Does anyone here have any experience on it? Any
> suggestions?
> Many thanks and have a nice day,
> Ann
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