Does anyone use the Phosphohistone H3 (Ser10) routinely in evaluating mitotic figures in formalin-fixed, paraffin-embedded tissues? I am using the antibody from Millipore which has been cited in the literature, but I get a lot of nonspecific nuclear staining besides mitotic figures and have exhausted my options such as high and low pH heat-induced antigen retrieval (EDTA @ pH9 vs citrate buffer at pH6), high dilution (up to 1:2000), use of a background reducing antibody diluent (DAKO), room temperature incubation vs. 4 degrees C, etc. I have followed Materials and Methods from the different citations but my samples are not as "crisp" and "clean" as the photographs in the papers. I am using archival samples and suspect that this is some of the problem. Am curious if anyone is using it in their laboratory or in the clinical setting and would be willing to share their protocol. I want to be able to use digital imaging analysis to assist in the quantification of the mitotic figures but not sure how I can with these non-G2 phase nuclei staining. Thanks in advance.
