[Histonet] Formalin Fixation times for IHC samples

Geoff McAuliffe mcauliff <@t> umdnj.edu
Wed Feb 16 16:39:34 CST 2005

Dear Anne:

    Standard answer, it depends on the antigen in question. Some survive 
fixation for 24 hours or more, some are rendered unreactive in 4 hours. 
Formalin works slowly so longer fixation usually reduces 
immunoreactivity. Also, the method of detection is important. In my 
hands the Vector ABC Elite kit gives a much stronger signal than the 
standard kit and often gives good results when the standard kit shows 


Anne C Lewin wrote:

> 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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>Histonet <@t> lists.utsouthwestern.edu

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu

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