At 09:26 11/05/00 -0700, Victoria Baker a écrit:
>Hi All!
>
>The foundation is looking into extracting proteins
>from paraffin/formalin fixed tissue samples.  Thus
>far, the success is eh to not so hot.  I was wondering
>if anyone had a procedure to do this, currently I
>haven't found anything that really gave me an answer.
>Given that the protein is masked by fixation, it seems
>logical that their has to be a method to unmask it.
>In IHC it's digestion, retrieval or both in a
>combination.  If anyone has a source I could tap into,
>I'd really appreciate it.

If you use formalin-fixed tissue, it's quite normal to have some problems.
The main problem is that -some of the proteins will be crosslinked, and
will thus not migrate at the correct MW in electrophoresis. Also, the
cross-linked proteins are difficult to extract. Better results can be
obtained using non-additive fixatives like Carnoy or methacarn. I have done
this a few years ago using 5% SDS to extract the dewaxed sections. In
SDS-PAGE you still have a smear, but many proteins will give an almost
correct appearance in Western Blots. If you want to have an idea on the
results that can be obtained check Journal of Neurocytology 23: 297-311. We
used this to check the specificity of anti-neurofilament monoclonals for
different species. As we had some trouble obtaining fresh tissue for
extraction, we used our paraffin embedded samples. This approach is also a
much better control than using fresh tissue, as the proteins extracted from
paraffin sections have gone to a complete histological processing, and are
thus somewhat closer to the antigen present in the tissue sections.

Paul
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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04  Fax. 03.88.24.04.61
========================klosen@neurochem.u-strasbg.fr==================3D=======
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