Re: your mail

From:"J. A. Kiernan" <>

On Thu, 25 Jan 2001, Su, Phy-Huynh wrote:

> trained to make 4% paraformaldehyde in PBS fresh to use. ...
> Can I make stock 10% in 2.5X PBS and keep at 4 degree for sometime?  If yes,
> how long can I store?  If no, what's the reason for not being able to do it
> and have to make it fresh?  Will the molecule cross link themselves and
> therefore reduce the fixative strength on tissues?

1. Freshly made "4% paraformaldehyde" doesn't contain any
   paraformaldehyde. (See recent Histonet discussion.)
2. The formaldehyde in the solution does re-polymerize slowly
   (months to a few years), leading to opalescence or a white 
3. Polymerization is faster at low temperatures, so there is
   no point keeping the solution cold unless you intend to
   use it cold (which will make it penetrate and fix more
   slowly than at room temperature).
4. The concentration of formaldehyde in a fixative is not
   a critical matter. The composition of the solvent (water,
   buffer salts, alcohol etc) makes much more difference to
   the specimen than the concentration of formaldehyde.
5. It is easier to make up neutral, buffered formaldehyde
   solutions from a bottle of formalin (which contains 37% by
   weight = 40% w/v) than from paraformaldehyde (with all the
   hoo-ha of heating it in a fume hood, waiting for it to cool
   down etc.) The solutions are almost identical, especially
   if the formalin is not taken from a bottle that's several
   years old. The difference (presence of about 1% methanol in
   a 4% solution made from formalin) has no effect on the
   fixative properties, even for subsequent electron microscopy.

These statements are all supported by plenty of published
literature, cited in textbooks and also in numerous Histonet
contributions from various people. A search of (the Histonet archives) should yield
plenty to read about formaldehyde. You might also like to
look at an article in Microscopy Today 00-1 pp 8-12 (Jan 2000),
which discusses these matters.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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