[Histonet] Glycerol cryoprotected tissue and cryostat sectioning

From:Kathie A Berghorn

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 Ám 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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