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Many years ago, we experienced the same difficulty from time to time in
using the acid-denaturation method for immunostaining of incorporated BrdU.
After repeated experiments, one of my colleagues (Dr. T. Murakami) noticed
that a real problem was a low pH (after neutralization). A tiny droplet of
HCL solution which remained at the top of the test tube came down and
lowered pH (to 2 or 3). At low pH, antibodies do not work well. We must be
very careful about it. Fill the neutralizing solution to the top of the
tube (or add it to overflow). This precaution solved similar problems of
many friends. I hope it solves your problem too.
Manabu Takahashi, M.D. & Ph.D.
Professor Emeritus in Yamaguchi University
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web