Hi!
Be sure to store your paraformaldehyde in glass as plastic will absorb.  (I use 4% unbuffered, stored @4C for not more than 1 week.)  Using for immunos and ISH.  Good luck!

-----Original Message-----
From: m.vermehren@t-online.de (Margarete Vermehren)
To: Goodwin Sylene <Sylene.Goodwin@UCHSC.edu>, histonet@pathology.swmed.edu
Sent: Thu, 30 Jan 2003 21:29:40 +0100
Subject: WG: 3%-4% Paraformaldehyde

This is an email I got concerning paraformaldehyde. I tried out the recipe -
it works fine.
Preparation of Paraformaldehyde Solutions

**Caution:  Preparation should be done in a chemical hood.**
Weigh out 40 grams of reagent grade paraformaldehyde powder and pour into a
2000 ml Erlenmyer flask.  Add distilled/deionized water to the flask to make
to one liter.  Add a stirring bar.   Heat the liquid to 70#186# with stirring.
Add 1 drop of 10M KOH or 10M NaOH.  The solution should become clear after
the addition of the base.  Allow the solution to cool to room temperature.
Adjust the pH to 7.3.  Add 50 ml of pH 7.3, 0.2M McIlvanies citrate buffer
per liter.  Use the fixative at room temperature.  Add 20 volumes of
fixative per volume of tissue.  Smaller tissue pieces (3mm thick or less)
fix better.



McIlvaine's Buffer (1921)
200ml mixtures of  x   ml 0.2M Na2HPO4 with   y   ml 0.1M citric acid

pH	x  ml  Na2HPO4	y  ml citric acid

7.2	173.9			26.1
The citrate buffer serves as a buffer against the formic acid that naturally
occurs over time in a formaldehyde solution.  If a buffered paraformaldehyde
solution is properly prepared it should store well for 3 months or more at
4#186#C.  However, it should always be warmed to a least room temperature, or
preferably 37#186#C, before placing the tissue specimens in it for fixation.
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 What I do now is this:

Here is how we recommend making PFA. (another recipe)

4% PFA (in 0.1M PB, phosphate buffer): 8g PFA dissolved in 100ml of dH2O.
Heat to water (on heated stirrer and stir using magnetic bead) to 55-60C to
partially dissolve PFA then add 1M NaOH dropwise (one or two drops) until
solution clears. Add 100ml of 0.2M PB, mix. Filter. Cool to 4C before use.
Store at 4C. Prolonged storage (>1 week) may require that pH is checked. pH
should be around 7.4, DO NOT autoclave!!

I then freeze aliquotes and use them that way - sure easy! By the way this
para also stores like the one above for 3 months at 4#176#C
Have fun,
Margaret


Margarete Vermehren
Willibaldstrasse 22B
D-80689 Munich
Germany
tel:  ++49 89 5801258
Fax:++ 49 89 5807298
Email: mvermehren@web.de

-----Urspr#252#ngliche Nachricht-----
Von: Goodwin Sylene [mailto:Sylene.Goodwin@UCHSC.edu]
Gesendet: Donnerstag, 30. Januar 2003 19:10
An: histonet@pathology.swmed.edu
Betreff: 3%-4% Paraformaldehyde

What is the general  rule for the refrigerator life of 4% paraformaldehyde?
Also, for IHC purposes on cells, are there preferences for the preparation?
i.e. heat versus pH?

thanks!

Sylene MJ Goodwin, B.S. HTL(ASCP)
Dept. of Gasteroenterology
University of Colorado Health Sciences Center
4200 E. 9th Ave
BRB 742/Campus Box B-158
Denver, CO 80262

(303)315-3520
FAX (303)315-3507