[Histonet] IHC on fresh frozen

Kim Merriam kmerriam2003 <@t> yahoo.com
Thu Dec 4 06:47:27 CST 2008

We use acetone/ethanol (as recommended by Gayle Callis in one of her NSH classes).  The slides look great; nice crisp nuclei (which you just won't get with acetone alone).  We air dry the slides and fix for 10 minutes in RT acetone/ethanol (75% acetone/25% absolute ethanol) and then put the slides into wash buffer and continue with IHC. 

I have heard that this fixative could be problematic with some antigens, but we have not found that to be the case with anything we have used in my lab.  Insulin should be fine.

 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 

From: Patti Loykasek <ploykasek <@t> phenopath.com>
To: "FU,DONGTAO" <fudo <@t> ufl.edu>; histonet <@t> lists.utsouthwestern.edu
Sent: Wednesday, December 3, 2008 11:38:07 AM
Subject: Re: [Histonet] IHC on fresh frozen

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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