[Histonet] IHC on fresh frozen

Sebree Linda A LSebree <@t> uwhealth.org
Wed Dec 3 10:12:51 CST 2008

I was taught to get frozen sections into cold acetone immediately after
sectioning, not letting the section air dry.  We do C4d staining on
frozen renal bxs and immediately fix the sections in cold acetone for 10
minutes.  Then we air dry followed by 2 minutes in Ventana Morphosave
(not a critical step) then keep the slides wet throughout the
immunostain.  We get nice clear, crisp staining.

Linda A. Sebree
University of Wisconsin Hospital & Clinics
IHC/ISH Laboratory
DB1-223 VAH
600 Highland Ave.
Madison, WI 53792

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Sent: Wednesday, December 03, 2008 9:17 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC on fresh frozen

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 

  Many thanks and have a nice day,


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