[Histonet] PFA preparation

[Roger Moretz]

I always prepared PFA as at least an 8% solution so that dilution with buffer would give me the final working %.  The protocol was to add the PFA powder to water at 50C and then add 10N NaOH dropwise until the solution cleared.  After cooling to RT, the stock PFA solution was filtered to remove any remaining insoluble particulates.  For a 4% PFA working fixative, mix the 8% 1:1 with a 2x buffer.  Osmolarity and buffer strength are correct in the final fixative.  Similarly, by adding dH2O one can easily formulate lower conecentrations of formaldehyde in the fixative.  Worked for years in an EM Lab where our standard fixative was 2% PFA and 1% glutaraldehyde.

Roger Moretz



----- Original Message ----
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
To: histonet <@t> lists.utsouthwestern.edu; ejschmid <@t> ucalgary.ca
Sent: Saturday, December 6, 2008 4:49:32 PM
Subject: Re: [Histonet] PFA preparation

You were taught correctly. And the socalled "degradation" is re-polymerization as you wrote. The only thing is that you should add buffer salts bettwen than PBS.
René J.

--- On Sat, 12/6/08, ejschmid <@t> ucalgary.ca <ejschmid <@t> ucalgary.ca> wrote:

From: ejschmid <@t> ucalgary.ca <ejschmid <@t> ucalgary.ca>
Subject: [Histonet] PFA preparation
To: histonet <@t> lists.utsouthwestern.edu
Date: Saturday, December 6, 2008, 3:30 PM

Hi,

  So then what is the best way to prepare formaldehyde fixative from PFA?

  The way I have been taught, which differs from what I have read, is to
dissolve 4% into ddH2O at room temperature. After that one could add
PBS or buffer.

  I've also been taught that too much heat during preparation
"degrades"
PFA, and that PFA (or formaldehyde solution, rather) stored too long
will lose freshness because it "degrades."

  What I read is different. Texts suggest to dissolve the PFA in warm
water, and that aging of the fix is due to repolymerization, not
through degradation.

What should i do?

Eric Schmidt

University of Calgary
Medical Sciences




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