Here is the protocol that I have used for years.  I have never had to
adjust the pH after making the paraformaldehyde and the procedure is
very straightforward.  I usually make 2% paraformaldehyde for my use,
but I have made 4% for others (following the same protocol) and they
have used it in their immuno protocol with very good results.  Note, the
final concentration of the phosphate buffer is 0.1M.

 

      0.2M phosphate buffer (stock)

A.    monobasic NaHPO4 (fw 137.99)        2.78 gm

      diH2O                               100 ml

 

B.    dibasic Na2HPO4 (fw 141.96)         28.4 gm

      diH2O                               1000 ml

 

mix 60 ml (A) and 920 ml (B).  Adjust pH to 7.4 store @ 4 C.  (very
little adjustment should be needed here based on these volumes)

 

 

      2% paraformaldehyde

 

A.    0.2M phosphate buffer               500 ml

B.    diH2O                               500 ml

C.    EM grade paraformaldehyde           20 gm

 

mix (A), (B), and (C), stir while heating to 60 C.  If remains cloudy,
adjust pH to 7.4 with 1N NaOH (I have never had to do this).  Store at 4
C.

 

      To make 4% just double the amount of paraformaldehyde.

 

 

 

Joseph A. Jurcisek

Senior Research Associate

Center for Microbial Pathogenesis

Columbus Children's Research Institute

700 Children's Dr.

Columbus, OH 43205-2696

(614) 355-3521

fax (614) 722-2818

jurcisej@ccri.net

 

 

-----Original Message-----
From: Hawkins, Hal K. [mailto:hhawkins@utmb.edu] 
Sent: Monday, July 24, 2006 3:44 PM
To: histonet
Subject: [Histonet] paraformaldehyde

 

 

 

We need to prepare 4% phosphate-buffered paraformaldehyde for a

cooperative study in fairly large volumes, about 2 L at a time.  

 

 

 

My only experience in preparing this fixative involved heating to over

60 C, adding sodium hydroxide chips until all was dissolved, then adding

the buffer and adjusting the pH.

 

 

 

There seems to be a simpler method in which pre-mixed Dulbecco PBS is

warmed and the paraformaldehyde is dissolved in the buffer.  Has anyone

used this method and does it work without further adjustment of the

final pH?

 

 

 

Thanks in advance,

 

 

 

 

 

Hal Hawkins

 

UTMB, Galveston, Texas

 

 

 

 

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