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

From:Thomas Mitchell

One step further with this note. For straight morphology, 24-48 hours  
of fixation in 10% formaldehyde followed by 70% ethanol, how long do  
you feel comfortable keeping the tissue in 70% ethanol before  
processing? If you canít process for whatever reason, any thoughts on  
what to keep tissues in until processing occurs?

Tom


On Oct 4, 2006, at 1:33 PM, Patsy Ruegg wrote:
" Fixation is generally carried on by the alcohols used for the  
processing.Ē
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  
can be reversible?

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


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