Dear Dr. Paul Zimmer 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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