[Histonet] Paraffine-embedded tissue

[Carmen Maria Garcia Pascual]

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