Re: [Histonet] Washing out formalin fixation (Lengthy)

From:"Bryan Hewlett"

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. Ltd.
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 
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" 
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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