[Histonet] Proof that fixation in formaldehyde solution can
bereversible?
Bryan Hewlett
bhewlett <@t> cogeco.ca
Tue Oct 3 13:32:32 CDT 2006
Rene,
Sorry to pursue the 'can of worms' further but;
The coefficient of infusibility for formaldehyde given in Bancroft is NOT based on Medewar's work, it is obtained from Baker (1958)
Baker based this on the work of Tellyesniczky(1926) who used thick tissue samples and long fixation times.
T. observed the visible changes and concluded a fixation time(wrongly, since he did not know that fixatives follow the laws of diffusion) in mm/hour.
Baker calculated a coefficient of 0.78 from this data.
Medewar(1941) proved that fixatives follow the laws of diffusion. He used plasma clots and stated a coefficient of 5.5 and commented that it was probably less, due to the nature of cell walls.
Baker repeated the experiments using gelatin/albumen gels to more closely mimic tissue. He quotes a coefficient of 3.6 and again points out that penetration in tissue would probably be less.
It is then that he mentions the data of Tellyesniczky and suggests 0.78.
The work of both Fox(1985) and of Helander(1994) using C14 labelled formaldehyde indicates that the K really MUST be at least 2.0 and most probably 3.5.
The numbness that you feel in your fingers after a few seconds is due to penetration of Methylene glycol not to formaldehyde binding!
Bryan
----- Original Message -----
From: Rene J Buesa
To: Bryan Hewlett ; GT Hebert ; histonet <@t> lists.utsouthwestern.edu
Sent: Tuesday, October 03, 2006 1:55 PM
Subject: Re: [Histonet] Proof that fixation in formaldehyde solution can bereversible?
Bryan:
This is I think the "can of worms" that was mentioned before.
If you put your fingers in formalin for just a few seconds you sill feel the numbness cause by the quick fixation and this same quick fixation is the one preventing the penetration with less and less penetration as more and more tissue is fixed.
The coefficients of diffusability for major fixing agents (Bancroft & Stevens: Theory and practice of histological techniques", Table 2.1 based on reserach by Medawar) are as follows:
potassium dichromate ---1.33
acetic acid ----1.2
ethanol---1.0
mercuric chloride--0.78 to 0.84
formaldehyde---0.78
That is why the combination of some of these reagents are able to take advantage of the quick fixation by formalin with the high penetration rates of the others.
That is why to obtain a proper fixation with NBF it is required, not only at least 24 hours, but also why almost any processing protocol includes 1 or 2 initial station for a secondary fixation.
René J.
Bryan Hewlett <bhewlett <@t> cogeco.ca> wrote:
Rene,
You wrote;
>>Formalin is an extremely fast fixative but with a very slow rate of
>>penetration, ....<<
Actually, the reverse is the case!< BR>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.
See;
Fox CH et al.
Formaldehyde fixation.
J. Histochem. Cytochem. 1985; 33, 845-853.
Bryan
----- 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
bereversible?
> 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
> 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.
>
&g t; 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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