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

From:"Bryan Hewlett"


You wrote;
>>Formalin is an extremely fast fixative but with a very slow rate of 
>>penetration, ....<<

Actually, the reverse is the case!
Formaldehyde has very fast penetration(possibly the fastest that we use) 
with a diffusion coefficient(K) of  at least 2.0 and most likely 3.5.
Compared to alcohol with a diffusion coefficient(K) of 0.8-1.0.
Penetration can be calculated from the following; d = K times the square 
root of time, where d = distance in mm and time is in hours.
However, the real problem is fixation. This is very slow, taking at least 24 
hours at RT for 90% of formaldehyde binding to occur.
This is due to the 'clock' reaction exhibited by formaldehyde solutions.
Once the binding has occurred , then more cross-linking can proceed.

Fox CH et al.
Formaldehyde fixation.
J. Histochem. Cytochem. 1985; 33, 845-853.


----- Original Message ----- 
From: "Rene J Buesa" 
To: "GT Hebert" ; 

Sent: Tuesday, October 03, 2006 1:04 PM
Subject: Re: [Histonet] Proof that fixation in formaldehyde solution can 

> GTH:
>  A piece of tissue sitting in PBS for 1 month after being fixed for 24-48 
> hours only is likely to have suffered tissue decomposition at the center 
> of it, specially if it was a thick piece. Formalin is an extremely fast=20
> fixative but with a very slow rate of penetration, this would have 
> determined external fixation but not complete fixation.
>  Fixation is not really reversible in a strict way and that is why you 
> need a strong Heat Inducet Epitope Retrieval (HIER) before IHC to "undo" 
> or "unfix" the crosslinkage produced by formalin.
>  The damage to the tissue could have been caused by improper storage = PBS 
> at room temperature during 1 month. If processed that tissue is very 
> likely to present altered microscopic appearance.
>  Just my opinion!
>  René J.
> GT Hebert  wrote:
>  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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