[Histonet] MART-1 staining

From:"Ingles Claire"

Greetings all:
I have recently been the lucky one to be given the task of trying to make MART-1 staining in our lab a reality. (or try anyway) I have all the protocols down for the most part. The only question I have is if I have to do any kind of enzyme digestion to open up the epitopes, etc and if so what kind. I am working on frozen skin sections. They are frozen in the cryostat and cut as soon as the tissue is brought into the lab. We are a Mohs lab, so the tissue hasn't been off the patient more than 10 minutes when we get it. I am currently trying out the Biocare prediluted MART-1, with the AP kit using Vulcan Fast Red (permanent) as a chromogen. I pH'd the buffer too before using and it was within acceptable range. So far there has been no specific staining, and only a little of what I would call background or non-specific staining along the basal layer where most of the melanocytes are located. The staining stains the entire basal layer, not individual areas where the known Melanoma areas are located. I have also tried the Innovex HMB-45 antibody with AP and fast red chromogen (aqueous) with even less of a reaction. Those sections are completely clean of any staining. Please help. I would also appreciate any other suggestions you may have.
Thanks a bunch,
Claire Ingles
Mohs Clinic
UW Madison, WI

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