[Histonet] paraformaldehyde
Jurcisek, Joseph
JurciseJ <@t> pediatrics.ohio-state.edu
Tue Jul 25 07:28:53 CDT 2006
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 <@t> ccri.net
-----Original Message-----
From: Hawkins, Hal K. [mailto:hhawkins <@t> 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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