Antibody-Based Technologies => Tumor Markers Review => Topic started by: DCundiff on November 03, 2011, 01:04:04 PM

Title: Phosphohistone H3
Post by: DCundiff on November 03, 2011, 01:04:04 PM
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. 
Title: Re: Phosphohistone H3
Post by: gula on November 04, 2011, 12:16:46 PM
We implemented PHH3 from Cellmarque on the Ventana Ultra. I have the data not at hand, but as far as I remember it was high pH retrieval 30 min and incubation for 32 min / 37C  with 1:100. Gave clear results.
Title: Re: Phosphohistone H3
Post by: hardrob on November 10, 2011, 10:01:16 AM
i use the p-histone 3 antibody by ,,Zytomed systems'' adressed here

it works really well on mouse, rat and human tissue. i tried several pretreatments like 2 min tapwater in the  microwave; the same with citrate- buffer/MW; pressure cooker, enzymes and many more. they all worked real good, also on decalcified bone tissue or bouin fixed testis.i'm trying to add a pic to this....