[Histonet] help with MART-1 on frozen sections

From:Constance McManus

Histo Landers!  Hi!  

It's been over a  year since I was on this list.  I
have had some very wild and hairy adventures setting
up a business and now I'm back doing histology in a
Moh's clinic at the University of Utah.  I find this
very exciting and  really like it.  It's waaaaay
different from the veterinary stuff was doing for such
a lont time.  Anyway, I'm glad to be back. 

One of the docs wants to set up MART-1 for melanoma.
If anyone out there is doing this on frozen sections,
I would very much appreciate your advice on the
questions below and anything else you might think of.

1) What do you fix your sections in?  We routinely fix
in acetone and I assume acetone is acceptable for IHC,
also.  Please verify if this is OK

2) I assume, but am seeking verification, that either
a nonsense ab or buffer need to be applied to the test
tissue and the pos ctrl tissue for a negative ctrl.

3)  what do you rinse with?  My procedure says to
rinse with TRIS, but no mention of any surfactant
(Triton X or Tween 20, etc).  I'm inclined to add the
surfactant, but would like verification if this is OK
on frozens.

4)  Who do you recommend purchasing ab's and
ancilliary reagents from? Are there any RTU ab's out
there that are reliable?  

5)  I have never worked with biotinylated abs before,
but it seems to me they would eliminate the linking ab
step, thus making the staining time shorter. 
thoughts??  What are some things to be aware of when
using biotinylated abs? 

I think that's all for now.  
thanks bunches!
Connie McManus HT

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