[Histonet] Glycerol cryoprotected tissue and cryostat sectioning
Dear Histo-netters,
I need your help. We have precious tissue that
has been processed for beta-galactosidase. This
was the first time we did this
immunohistochemistry. The protocol said to clear
in glycerol which we did and the tissue is in 80%
glycerol. I have spent the morning trying to cut
this tissue on a cryostat without success. When
I put the mouse placenta in OCT using isopentane
and liquid nitrogen, the OCT freezes but the
tissue does not. Cutting it on the cryostat is
then not possible. When I used to do thick
sections we sunk tissue in sucrose and then froze
it with dry ice prior to cutting it on a freezing
sliding microtome. Would the dry ice freeze the
placenta in the cryostat so I could cut it? Any
other suggestions? Of course I am on a grant
deadline and am stressing.
I would appreciate any suggestions that would
yield 8-10 µm sections so I can see if the beta
galactosidase worked in the area of the placenta
we study.
Thank you very much,
Kathie
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