[Histonet] Formalin Fixation & IHC

Bryan Hewlett bhewlett <@t> cogeco.ca
Wed Nov 1 14:55:03 CST 2006

Hi Helen,

"Overfixation" was a term used by early histologists to describe the 
unexpected and excessive hardening and shrinkage of tissues
which occurred following lengthy exposure to certain fixatives.
It gave rise to the concept of tolerant and intolerant fixatives.
Heidenhain's SUSA and Zenker's fluid are examples of intolerant fixatives 
and textbooks usually suggest that fixation times should not exceed 24 
On the other hand, an aqueous solution of formaldehyde is the classic 
example of a tolerant fixative,
tissues may be left in it for extended periods of time, with no excessive 
hardening or shrinkage.

So-called 'overfixation', as applied to fixation of tissues in aqueous 
solutions of formaldehyde for IHC, is a total misnomer.
Simply put, it never occurs in any clinically relevant time frame 

Formaldehyde fixes by formation of hydroxymethyl adducts on reactive side 
chains of proteins.
Once sufficient adducts are formed, they slowly cross-link to stabilize the 
proteins in a gel-like formation.
At room temperature, it takes approximately 24 hours for maximal binding of 
formaldehyde to occur and hence all the adducts to form.
These initial adducts, and any initial cross-links formed, are unstable and 
easily reversed.
For tissues fixed for 24 hours, the cross-links are largely reversed by 
washing ( in water or 70% EtOH) after a few  hours.
It probably takes at least 5-7 days for most of the cross-links to form, and 
a small amount of cross-linking still continues over time.
Even after fixation for 7 days or more, the cross-links can still be 
reversed by prolonged washing.
That's great for IHC, since cross-links can be reversed by HIER (which is 
just washing at elevated temperatures!).

We have tested over 300 antibodies, using human tissues fixed in NBF for 
times ranging from 2 hours to 3 months ( some out to 1 year).
In no case were we unable to demonstrate the antigen by IHC in tissues fixed 
for 24 hours or longer.
In contrast, many antigens (particularly surface proteins) failed to be 
demonstrated in the same tissues fixed for less than 24 hours!!!

Don't worry about so-called 'overfixation', there is NO SUCH THING.
Do worry about underfixation, it can be devastating to IHC.
My advice, leave the tissues in formaldehyde for up to 90 days and then if 
you must, transfer to 70% EtOH for further storage.



----- Original Message ----- 
From: "Helen E Johnson" <hej01 <@t> health.state.ny.us>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, November 01, 2006 11:38 AM
Subject: [Histonet] Formalin Fixation & IHC

> Hi Histonetters,
>      In order to avoid overfixation of specimens in Formalin when
> performing IHC , specimens are usually transferred to 70% EtOH at 24 hrs.
> Is there any adverse effects of antigen damage in long term storage in
> EtOH?  Will specimens get hard if kept long in EtOH?
>                                                                    Helen
> Johnson   (hej01 <@t> health.state.ny.us)
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
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