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

From:"Patsy Ruegg"

" Fixation is generally carried on by the alcohols used for the processing.
Here in lies the problem for many antigens "unintended consequences" you
might say.  If the sample is not adequately fixed-proteins protected by
crosslinking, the alcohols can destroy the protein of interest.  We need to
fix for a minimum of 24 hours to allow for crosslinking which will protect
the proteins (especially surface proteins) from processing so that they are
still there and can be accessed after unmasking the formalin fixation.  If
certain proteins are not protected from tissue processing they will be lost
forever and no amount of epitope retrieval will restore them.
Patsy 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Rittman,
Barry R
Sent: Tuesday, October 03, 2006 10:49 AM
To: GT Hebert; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Proof that fixation in formaldehyde solution
canbereversible?

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. 

Barry 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of GT Hebert
Sent: Tuesday, October 03, 2006 11:29 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Proof that fixation in formaldehyde solution can
bereversible?

Hello,
   
  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
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
samples?
   
  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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