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

From:"Bryan Hewlett"


Nobody is suggesting this as THE way to retrieve tissue antigenicity.
Just pointing out the mechanism of the effect in answer to the original 

Actually in the real world, it pretty much DOES work this way!
Everybody does it everyday when they short fix tissue in NBF and then 
process it.
The reversal occurs in the processing alcohols and then, for some proteins, 
something even worse happens, they re-fix in alcohol!
The shorter the fixation time in NBF, the more rapidly the reversal occurs 
and the more like alcohol fixation is the end result.
Alcohol fixation can result in the loss of up to 40% of tissue proteins.
If your protein of interest is one of them, this can mean that IHC 
demonstration of the affected protein is compromised and no further amount 
of HIER is helpful.
This combined mixed pattern of formaldehyde and alcohol fixation is 
responsible for 90% of IHC staining problems.



----- Original Message ----- 
From: "Mark Tarango" 
Sent: Saturday, March 08, 2008 8:00 PM
Subject: Re: [Histonet] Washing out formalin fixation (Lengthy)

I just want to point out again that this works poorly and wastes water... I
don't want anyone to get too excited and ditch the HIER buffers.  Nobody
does this in the real world.  It all goes back to what René said, "It
(pretty much) doesn't work that way."

On Tue, Mar 4, 2008 at 7:56 AM, Bryan Hewlett  wrote:

> 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
> temperatures.
> 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
> re-hydrate.
> 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!!
> References:
> 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,
> 845-853
> 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.
> 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:
> 190-199
> Best regards,
> Bryan
> ----- Original Message -----
> From: "Johnson, Teri" 
> To: 
> 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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