Re: [Histonet] Proof that fixation in formaldehyde solution canbereversible?

From:"Bryan Hewlett"

Barry is quite correct.


Even after fixation times of longer than one week, formaldehyde fixation can 
still be partially reversed.
This is, after all, why HIER works so well.

Following fixation for 24-48 hours, sitting in PBS for a month will 
certainly have largely reversed the initial fixation.
There is evidence that following 24 hour fixation in formaldehyde a water=20
wash for 6 days will reverse 90% of formaldehyde binding. (see reference).
The tissues will have been largely re-fixed in the processing alcohols.
The effect of this will depend on the antigen of interest.
It may be beneficial (antigenicity restored) or detrimental.

The following reference may be of use;

Helander KG.
Kinetic studies of formaldehyde binding in tissue.
Biotechnique and Histochemistry. 1994; 69, 177-179.


----- Original Message ----- 
From: "Rittman, Barry R" 
To: "GT Hebert" ; 

Sent: Tuesday, October 03, 2006 12:48 PM
Subject: RE: [Histonet] Proof that fixation in formaldehyde solution 

This is really opening a barrel of worms but...

Re formaldehyde solutions.
If tissues are fixed for a few hours in standard formalin solutions then it 
is possible to reverse the fixation process by washing. It is a reversible 
process in that the bonds that have been formed are generally temporary and 
can be broken.
Technically if you wish for "adequate or true fixation" then Pearse provides 
the theory for why this should be for about 1 week.
(The fixation process does in fact continue for years).

Of course no one fixes for one week except by accident. However the fixation 
for 24 to 48 hours is only an initial fixation. Fixation is generally 
carried on by the alcohols used for the processing.

A. G. E. Pearse "Histochemistry Theoretical and Applied: volume 1 
Preparative and Optical Technology."
Churchill Livingstone.


-----Original Message-----
[] On Behalf Of GT Hebert
Sent: Tuesday, October 03, 2006 11:29 AM
Subject: [Histonet] Proof that fixation in formaldehyde solution can 


  I am in a heated debate with an investigator regarding his samples.  They 
were shipped to me after (24-48hrs fixation - 10% NBF or 4% PF unsure 
exactly which one) and then switch to PBS for shipment.  They sat at room=20
temperature for over 1 month before being processed routinely and embedded 
in paraffin wax.

  Can someone tell me if indeed the fixation is reversible, or once they 
have been fixed for over 24hrs they remain fixed??  Can anyone refer me to 
books or papers that talk about this??

  Also, what affect on antigenicity will such storage in PBS have on these 

  Thank you all so much for your help.

  G. H.
  Cambridge, MA

  Key words:
  NBF, Alcohol, 10%, paraformaldehyde, 4%, fixation, fix, ethanol, 70%, 
reverse, reversible.

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