[Histonet] Formalin Fixation times for IHC samples

Katri Tuomala katri <@t> cogeco.ca
Wed Feb 16 20:22:38 CST 2005

I think you are doing well with your protocol. Although I don't think 
"overfixation" is as a big problem any more with the advent of different 
HIER protocols with various buffers being used. Under fixation (less than 24 
hours) can be a bigger problem with certain antibodies like Her-2, ER and PR 
to mention few. This concept of over fixation is a remnant from the time 
prior to HIER, when we all had a problem trying to demonstrate many antigens 
with weaker retrievals (proteolytic enzymes). The heat over 60C was actually 
banned from any immunohistochemistry protocols 20 years ago.
My two cents worth...


Katri Tuomala
Hamilton, Ontario, Canada

----- Original Message ----- 
From: "Anne C Lewin" <anne.lewin <@t> bms.com>
To: <Histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, February 15, 2005 10:28 AM
Subject: [Histonet] Formalin Fixation times for IHC samples

> Could I get a general concensus from Histoland about the amount of time
> needed to fix a sample for IHC without over-fixing?  My training/journal
> searches  have led  me to believe that the longer a sample is in
> formalin, the stronger the protiens are cross-linked, and the more
> difficult it is to break those bonds with antigen retreival and get your
> antibody to the target.  I generally tell the scientists in my
> department to fix overnight (most samples vary from about the size of a
> pea to a lima bean), and then to transfer to 70% Ethanol for me to
> process for paraffin.  The total time in fixative tends to be around 24
> hours.  Morphology has always been great, and my IHC's work well.  This
> question is mainly for my information, since I have used the same tissue
> prep protocol for a few years now, I want to make sure I am keeping up
> with current opinions.
> Thanks!
> -Anne


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