Re: paraformaldehyde/PBS

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From:"Jacques Paysan" <paysan@Uni-Hohenheim.DE>
To:"HistoNet Server" <>
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I'm a bit surprised to see that dissolving paraformaldehyde in PBS seems to
be common practice.  I'm not sure if I understand the rational behind this
(I'm ready to learn though).  First, PBS (as 10 mM phosphate buffer with
salts) seems to be a weak buffering system if you consider that the fixation
process quantitatively produces protons.  We consider 150 mM phosphate
buffer to be a more reasonable concentration.  Second, a 4% formaldehyde
solution on its own should be highly hyperosmotic ( about 1300 mOsm if I
remember correctly) thus making the addition of salts rather a problem than
useful.  Third, why buffering the water in which the paraformaldehyde is to
be dissolved, then making it basic by the addition of (obviously large
amounts of) sodium hydroxide, and finally retitrating it back to neutral?  I
find it more practical to dissolve the paraformaldehyde in water (containing
about 0.01 N NaOH), prior to adjusting the pH with the concentrated buffer.

I have never heard that in this case the addition of whatever x-fold
concentrated buffer would precipitate formaldehyde from its aqueous
solution.  Neither have I ever heard that paraformaldeyde doesn't go into
solution with slightly basic water at 60C (with the exception of John
Kiernan's previous post about some strange paraformaldehyde subspecies).

The protocol from my previous post always worked, no matter what buffer we

Phosphate-bufferd formaldehyde.  Per 100ml:

70 ml distilled water
4 g paraformaldehyde ( we use the granular stuff sold by EMS because it's
easier to handle - no dust )
500 l 1 N NaOH
Warm in a 60 C waterbath for serveral minutes.
When everything is dissolved (usually < 5 minutes) add 30 ml 500 mM sodium
phosphate buffer pH 7.4.
Filter and cool on ice.


----- Original Message -----
From: "Tamara Howard" <>
To: <>; "Histology listserver"
Sent: Wednesday, May 24, 2000 2:19 PM
Subject: RE: paraformaldehyde/PBS

> Saw your post on the Histonet - which PBS are you using? This is
> nightmarish.....PFA takes *years* (OK - slight exaggeration) to go into
> solution in - for example - Dulbecco's PBS. Are you making up your buffer
> from scratch, or buying 10x from Gibco or someone? D-PBS has extra salts
> in it which prevent PFA from going into solution - we discovered this the
> hard way. Nothing wrong with the commercial PBS, just that this particular
> formulation is "bad" for PFA. Which, of course, brings up the question of
> which of the million PBS recipes that are out there do you use? We use the
> plain old sodium phosphates/sodium chloride mix; Ca/Mg-free.
> Anyway - if your PBS is pH'd to above ~6.8 before you dump in the PFA and
> start heating, you should be fine. pH 10 is out of control - unless you
> are using the fix at that pH.
> Tamara Howard

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