Dear Todd

we are looking for the expression of c-myc in colon cancer. The colon
cancer  specimens are already paraffinized. We get them from several
institutes of surgical pathology in Switzerland. We tried three antibodies
already mentioned in my first email (clone 9E10 (Santa Cruz), 9E10.3
(Neomarkers), A14 (Santa Cruz)). To evaluate the best working condition for
those antibodies on paraffin sections we applied a protocol for antigen
retrieval established in our lab. Testtissue consists of normal colonic
tissue. We found out that with antigen retrieval we yield a sufficient
result for the first two antibodies mentioned above: The staining is found
to be prominent in the nuclei of crypt cells in the colonic tissue
reflecting the proliferating pool of epithelial cells. In the upper part of
the crypt no nuclear staining was observed. Additionally, we found
prominent cytoplasmic staining of almost all epithelial cells, mostly
strong and not weak. Dilution of the first antibody diminished this
cytoplasmic staining, unfortunately also the nuclear staining.

According to a paper of Mona Melhem ( Distribution of cells expressing myc
proteins in human colorectal epithelium, polyps, and malignant tumors,
Cancer Res. 1992 Nov 1;52(21):5853-64), where she showed IHC in colonic
tissue using different self made antibodies recognizing c-myc, only one
antibodie had some cytoplasmic staing aside the nuclear staining. All the
others show only nuclear staining. This is true for normal colonoc tissue.
She tested aswell polyps and cancers and there she showed a clear
cytoplamsic staing aside the nuclear staining. Maybe due to a higher
production of c-myc in cancer than compared to normal colon.

IHC carried out on polyps and cancers yielded no result. We concluded that
it might be a problem of fixation because whenever we had the opportunity
to get the tissue fresh and we took care of the fixation, some staining was
visible. With most of the specimens of the pathologies we have no result.

Which antibody did you use for your investigations and under which
condition they worked best? Do you have any suggestions, how to address
this probem?

Thank you very much

Michael



Michael Stumm, MD, Lab 405
Department of Research
Kantonsspital Basel
Hebelstrasse 20
4031 Basel
Switzerland
phone: ++41/61/265 23 84
email: stumm@ubaclu.unibas.ch