Constance McManus ladylaynah <@t> yahoo.com
Wed Jul 11 20:47:12 CDT 2007

Well, gang, I posted a question some time ago about
mysteriously skipping staining of MiTF IHC frozen
sections.  Some of you responded with some really good
information, but no one could tell me why the stain
skipped on some tissue and stained other tissue in the
same block.  I figured it out myself as I was checking
my e-mail over breakfast one morning.  In a word or
two -- freeze-thaw and freeze again.  During our
embedding  we were freezing the tissue to get the
epidermis flat and as we worked the next piece of
tissue, the first one thawed.  Then we froze both
tissues which caused freeze artifact.  This is deadly
to epitopes.  I have always taken great care to keep
our control tissues frozen (to the extent that I keep
them encapsulated in OCT and carry them to the ultra
low freezer in a LN2 cooled container) and I store the
cut control slides in a -20C freezer, taking out only
what I need.  But I never noticed the freeze/thaw
going on as we embedded our tissues.  

What can I say, except that I feel so much like a
newbie.  Anyway, thanks for the responses.  

Connie McManus, HT
University of Utah
School of Medicine
Dept. of Dermatology

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