Re: [Histonet] MART-1 staining

From:Constance McManus

I am doing the same thing in our Mohs lab, except that
we are using MiTF (microphthalmia transcription factor
from LabVision - NeoMarkers) antibody insteadof MART-1
because my doc's lit search showed that MART-1 tends
to stain more promiscuously leading to ambiguity in
the interpretation. 

 Since you are using unfixed tissue, epitope retrieval
is not needed.  If you did an epitope retrieval step,
that is one possibility to explain the staining
problems.  Epitope retrieval is used on fixed tissues.

Did you optimize the antibody dilution?  Did you block
for catalase and protein?  These may be some other
causes of your problems. I was told to use HRP rather
than AP on this assay,  I did not get any good
explanation why .  Skin should be fine with AP, but
maybe it has something to do with the antibody itself.

Also, you didn't mention your sections.  Everything I
have read says that the frozen sections need to be
nearly perfect and cut between 2 -4 microns, the
thinner the better.   

If you want my protocol, email me and i will be happy
to share.

Connie McManus, HT
University of Utah School of Medicine

--- Ingles Claire  wrote:

> 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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