I am posting this question for a friend: I am testing the anti-caspase3 antibody that was sent to me. When I stained cells with the Ab, I see background staining even from the non apoptotic cells. In this experiment, I did not include isotype control. So, re-test with isotype control is neccessary. Still, I have several qeustions regarding intracytoplasmic staining. 1. I used permiabilizing soln from BD. Have you used it before? 2. For washing, I just used PBS. Pharmingen protocol suggests to use their permiabilizing/wash buffer. What do I have to use for wash? 3. Do I have to fix them? Although the protocols from BD and Pharmingen suggested to fix them, I did not because I thought it is not much different from the surface staining. We would appreciate any suggestions! Thanks! Stacie Stacie Anderson Laboratory of Gene Transfer, Hematopoeisis Section NCHGR, NIH Room 2C12 49 Convent Drive Bethesda, MD 20892-4442
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