[Histonet] Washing out formalin fixation (Lengthy)

Bryan Hewlett bhewlett <@t> cogeco.ca
Tue Mar 4 11:55:19 CST 2008

Hi Tim,

Thanks, I am aware of both papers and also a follow-up discussion at BSC 
last year.
The authors also indicate that some epitopes do not mask or require HIER to 
demonstrate, even though they have been 'fixed' for long enough.
This is of course born out by every day experience in the IHC lab.
Interestingly, they also point out that some peptides that are fixed this 
way and do not require HIER, WILL require HIER if fixed in the presence of 
another protein or peptide.
That of course is also the case in the real world of tissues!!!

(See also Yang K, Sompuram SR, Fitzgibbons P Bogen SA. National HER2 
proficiency test results using standardized quantitative controls: 
characterization of laboratory failures.
Arch Pathol Lab Med 112, February 2008 pp 211-216.)

As you know, I have long decried the practice of short fixing small biopsies 
and the effects of reversal and refixation by alcohol during processing.
This can be particularly devastating on surface proteins such as HER2 etc. 
The 6 hour minimum fixation time in the HER2 guidelines is, in my opinion, 
OK, I'll step off the soap box.



----- Original Message ----- 
From: "Morken, Tim" <tim.morken <@t> thermofisher.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Tuesday, March 04, 2008 12:16 PM
Subject: RE: [Histonet] Washing out formalin fixation (Lengthy)

Thanks for the synopsis Bryan.

I'd like to point out that the 2004 paper by Sompuram,and a followup in
2006 are very interesting. These studies these show that fixation of
peptide spots (essentially zero thickness) attached to glass slides take
over 6 hours to "fix" at room temperature. In this case they defined
"fixed" as the point at which an antibody  would no longer detect it's
target epitope, or the reaction was severly diminished. They also showed
that HIER reverses the "fixation." This calls into question any method
that relies on less than six hours formalin fixation at room temperature
(ie, biospies, just because they are small) and also the effect of
exposing those short-fixed tissues to  long exposure to 70% alcohol, or
other aqueous solution, before clearing and embedding.

The 2006 paper also investigates why some epitopes are affected by
formalin and others are not.

Sompuram SR, Vani K, Messana E and Bogen SA. A molecular mechanism of
formalin fixation and antigen retrieval. Am J Clin Pathol (2004);121:

Sompuram, et. al, A molecular model of antigen retrieval using a peptide
array, Am J Clin Path 2006;125:91-98

Tim Morken
Technical Support Manager
Lab Vision Products
Anatomical Pathology
ThermoFisher Scientific

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Bryan
Sent: Tuesday, March 04, 2008 7:56 AM
To: Johnson, Teri; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Washing out formalin fixation (Lengthy)

Hi Teri and everyone else on this thread,

Washing out many of the effects of formaldehyde fixation on tissues with
running water has been known for years (much longer than this old boy
has been around).
In modern terms, it is the essential underlying mechanism for so-called
antigen retrieval (HIER).

Formaldehyde fixes proteins by addition, with the formation of
hydroxymethyl adducts on the reactive side chains of proteins.
Once enough of these hydroxymethyl adducts are formed, and IF they are
in close approximation to each other, they may slowly cross-link by the
formation of methylene bridges.
However, these adducts and initial cross-links are unstable and readily
reversed by water and alcohol (see Kiernan (1999).
It takes 24 hours at room temperature for all the hydroxymethyl adducts
to form, i.e. maximal binding threshold (see Fox et al, 1985).
If the tissue is then exposed to running water before all the adducts
have formed (i.e. less than 24 hours), the reversal is very rapid.
The shorter the time in formaldehyde, the more rapid the reversal.
Even after the essential 24 hours fixation and also after a more lengthy
6 days fixation, running water will still remove the adducts and
hydrolyse the methylene bridges.
There is at least one publication (Helander, 1994) that provides data
regarding this effect.
After 24 hours fixation, 50% reversal occurred in less than 24 hours,
90% reversal was obtained after 6 days washing and for 6 days fixation
90% reversal after 4 weeks washing.
It should be noted that these reversal times were obtained at ambient
temperatures and the times may be considerably reduced by elevated

This reversal effect is also obtained on tissue sections that have been
processed to wax.
However, because of the additional shrinkage and hydrophobicity of the
processed proteins, the reversal is slowed somewhat until the proteins
The reversal effect can also be aided by the presence of other ions in
the water (the purpose of HIER buffers).
Back in the sixties, in order to successfully demonstrate Ig's by IF, we
were reversing the fixation effects on paraffin sections by placing them
in hypotonic buffers for 2 days at 37C.
Today, since we are all in a great rush for results, we obviously drive
the reversal at higher temperatures to speed things up!!


Baker JR (1958), Principles of Biological Microtechnique, Methuen & Co.
Hopwood D. Fixatives and fixation: A review. Histochemical journal
(1969); 1, p19-55 Burnett MG. The mechanism of the formaldehyde clock
reaction: Methylene glycol dehydration. J Chem. educ. (1982); 59, 160
Fox CH et al. Formaldehyde fixation. J Histochem Cytochem (1985); 33,
Helander KG. Kinetic studies of formaldehyde binding in tissue.
Biotechnique and Histochemistry. (1994); 69, 177-179 Kiernan J.A.,
Histological and Histochemical Methods: Theory and Practice, 3rd
Edition, (1999). Oxford: Butterworth-Heinemann. ISBN # 0-7506-3106-6.
Shi SR, Gu J. and Taylor CR. Antigen Retrieval Techniques:
Immunohistochemistry and Molecular Morphology. (2000) Eaton Publishing.
ISBN 1-881299-43-0.
Sompuram SR, Vani K, Messana E and Bogen SA. A molecular mechanism of
formalin fixation and antigen retrieval. Am J Clin Pathol (2004);121:

Best regards,


----- Original Message -----
From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, March 03, 2008 2:33 PM
Subject: [Histonet] Washing out formalin fixation

Last week, a researcher here asked me what the chemical mechanism was of

washing out the effects of formalin fixation on the tissues with running

water. In other words, how does it work? Anybody here know?

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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