Re: Fixative Buffering

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From:Geoff McAuliffe <mcauliff@UMDNJ.EDU>
To:Jacques Paysan <paysan@Uni-Hohenheim.DE>
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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).

    Well, I am no chemist so I can't expound on aspects of buffering but I do
use paraformaldehyde at high pH, about 10.6 for IHC fixation. I use sodium
phosphate dibasic with a enough NaOH thrown in to raise the pH to the desired
level. Crude, but it works very well. Are you familar with familiar with Berod
et al. J. Histochem. Cytochem 29:844-850, 1981? They consider the effects of
high pH fixatives on tissue.


Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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