paraformaldehyde fix

Richard Kerr kerrr at CRYPTIC.RCH.UNIMELB.EDU.AU
Wed Sep 24 21:38:54 EST 1997


I may be able to shed some light on this question.

paraformaldehyde powder is a reasonably stable polymer. when you dissolve it
in neutral or basic solutions, it depolymerises to become formaldehyde.  If
kept for some period at RT or in contact with oxygen, the aldehyde moiety
undergoes oxidation to formic acid (aldehyde to carboxylic acid...chem 101)
or a ketone (???)
As PFA is used for immunocytochem to preserve both tissue and epitopes
within the tissue, any other chemicals (like formic acid) bring variables
into the 'fixation equation'as well as altering the pH of the fixative.
Thus, for consistancy, the dogma is ..fresh every time.
Of course, all this worry is avoided if you KNOW that your epitope of
interest is not formic acid or ketone labile.
further, the 'routine grade' formalin that the histo lab will use is
formaldehyde in a neutral buffer with up to 10% methanol added as a
'preservative', possibly to stop the conversion to formic acid.

Tied up in this wedge of text are hints and reasons to explain why some
people have no trouble with this issue ( David's "I keep mine in the fridge
in the dark and its fine" contribution; lucky fellow) and why its the bane
of other people's projects.
Personally I make a 4 % PFA stock in PBS, aliquot it and freeze it.  When I
need some I thaw out a tube, filter it to remove crystals and use it.  Just
my $0.05 (they don't have $0.02 here anymore)

Richard


At 14:29 24/09/97 -0700, you wrote:
>At 15:57 9/24/97 -0400, Stan A. Baldwin wrote:
>>I've had several people tell me that paraformaldehyde made in solution
>>is no good after a couple of days.  I've used month old paraformaldehyde
>>from the frig and it fixes tissue just fine for immunocytochemistry and
>>general histology.  Does anyone know if there is some chemistry reason
>>why para would "go bad" after a couple of days in the frig.
>
>Steve:
>
>I too am interested in this.  I've always made a 2% stock of PF and kept
>that dark and in the fridge.  I dilute what I need with PBS to a final of
>1% PF.  I've kept the stock for months (like.. 6 months..).  In flow
>cytometery, fixation alters the FALS and 90'LS of cells and drops them a
>bit.  This alteration has not changed with time and I've seen no
>significant effect on FITC signal over the months.  If PF has a very short
>shelf life, then I figure that I've been very lucky.  I've gone through
>about 6 batches of PF and what determines when I need to make more is when
>I run out.
>
>I'd be interested in other comments you receive.
>
>David
>
>
>
Richard Kerr.
The Murdoch Institute,
R.C.H. Flemington Rd, Parkville, 3052,
AUSTRALIA.
kerrr at cryptic.rch.unimelb.edu.au
Phone (61) 3 9345 5045.
FAX   (61) 3 9348 1391.
'The most interesting things about vertebrates occur in the neural crest.'
	Peter Thorogood.




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