Hello everyone: I am Hortensia from the University of Valencia, from Spain. I am new in this world. And I have some issues. I will apreciatte if someone with experience can help me. I am working with endometrial tissue samples, from biopses and endometrial aspirates. I want to detect the nervous fibres with 9.5PGP antibody (from DAKO). I am using as positive control large intestine, and I am getting a very clear staining. But when I perform the immunohistochemistry in the endometrial samples I don't obtain any staining. so, I think that the problem may be in the previous steps, like the pick of the sample, the fixation and/or the paraffine incluison protocol. I fix the samples 12-24hs in formaline 10%, the paraffine inclusion protocol is the next: etoh 70%, 96% and 100% (2 changes 1 hr each); xilene 2 changes x 30 min and 1,5hr twice in paraffine, then I cut the sample. I want to know if this protocol is correct and if the samples are very small, should I decreased the incubations times at the half? In summarize, which is your paraffine inclusion protocol? and this protocol is independent of the sample size? Thank you very much!!!!! Best regards, Horten
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