Re: Fixative Buffering

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From:"Tim Morken" <tmorken@mailcity.com>
To:histonet@pathology.swmed.edu
Reply-To:
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>Jacques wrote: 
>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 ?


CalBioChem has a good booklet explaining various buffers and their uses. You can request it from their web site:

http://www.calbiochem.com/literature/Booklets.htm
---
Tim Morken, B.A. EMT(MSA), HTL(ASCP)
MS-G32
Centers for Disease Control and Prevention
1600 Clifton Road
Atlanta, GA 30333

PH: 404-639-3964
FAX: 404-639-3043

other email: tim9@cdc.gov



On Sat, 20 May 2000 18:23:06   Jacques Paysan wrote:
>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
>
>
>


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