[Histonet] Formalin Fixation & IHC
Hi Brian and Helen,
I agree with Brian to a limited extent. The range of antibodies that are
used in clinical diagnosis have been selected to work used on well fixed
antigens and tissues. I used them when available. However if you are
developing an IH reaction with a new antibody or using frozen sections the
antigens in the tissue can be very sensitive to the amount of fixation.
For example: I have one very tricky antigen the Dopamine transporter in the
brain. If you do a simple test with 4 sections: 1) no fixation; 2) 10 min
fixation, 3) 4 hour fixation or 4) overnight fixation. You will be able
to visualize the reaction only on the non fixed and lightly fixed tissue.
I do no clinical work only research work and use different types and levels
of fixation for each antibody! The answer is; 1) the amount of fixation
depends on how the antigen reacts to fixation and then if the antibody will
recognize the fixed form of the epitope on the antigen or not. 2) copy an
established method for that antibody. Usually get them from a friend, the
methods in specification sheets that come with the AB only work sometime.
Best of Luck
Assoc. Professor David Finkelstein,
The Mental Health Research Institute of Victoria,
155 Oak Street, Parkville, Victoria 3052
Date: Wed, 1 Nov 2006 15:55:03 -0500
From: "Bryan Hewlett"
Subject: Re: [Histonet] Formalin Fixation & IHC
To: , "Helen E Johnson"
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"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
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"
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?
> Johnson (firstname.lastname@example.org)
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