Dear Histonetters

In theory, formaldehyde should mainly react with unprotonated amino- and
guanidyl-groups of tissue proteins.  Since the degree of protonation of
these groups for a particular protein depends on the pH during fixation the
fixative buffering should be an interesting parameter to control the
fixation process, particularly if for example an epitope is fixation
sensitive.  Recently, I found a publication in Nucl. Acid Res. by Basyuk et
al (28: e46, 2000) where an alkaline fixation procedure was described,
improving in situ hybridization signals dramatically when fixing at pH 9.5.
We tested this and got similar results.  However, it appeared to us that
fixing in PBS at pH 9.5 sounds somewhat unreasonable because PBS should have
almost no buffering capacity at this range.  We used carbonate buffer
insted, which worked well, but we would like to extent this study to other
pH-values.  Since I'm not much into buffer chemistry I have the following
questions:

Can anyone recommend buffer substances with a variety of different pKa's
which are compatible with formaldehyde fixation ?  Is there a publication
available reviewing this ?  Which buffer substances are not compatible with
formaldehyde ? (I always thought that Tris-Buffers don't work because they
react with formaldehyde, but by a biochemist I was told that this is a myth
because Tris is a ternary amine and therefore will not react with the
fixative).

Anyway, I just ordered John Kiernan's latest Histology edition, hoping to
find some information in there.  But Amazon takes up to 8 weeks to deliver
and I'm affraid I need the information earlier.

I would therefore greatly appreciate all hints and suggestions.

Thanks,
Jacques

---------------------------------------------------------------
Jacques Paysan, PhD
University of Hohenheim
Institute of Physiology 230
Garbenstrasse 30
D-70593 Stuttgart
Germany

Phone: ++49-711-459.22.67
Fax:  ++49-711-459.37.26
email:  paysan@uni-hohenheim.de
Web:  http://www.paysan.de/lynx.htm