[Histonet] Cryosectioning cryoprotected (30% sucrose) brain

[Ben]

Hello All,

 

I am attempting to slice sheep brainstem and hypothalamus on a cryostat at
-20 C.  The tissue was treated as such:

 

Perfusion through carotid artery (6L of 4% paraformaldehyde)

Postfixation for 24 hours at 4 C

Cryoprotected in a 20% sucrose solution for 5 days at 4 C

Cryoprotected in a 30% sucrose 30% ethylene glycol solution and stored at
-20 C.

 

I am having some major problems with slicing this tissue.  It appears to
never freeze.  Does anyone know at what temperature I should be slicing?
Any opinions on use of a vibratome instead of a cryostat?  I'm trying to
section tissue at 30-40 um.   Thanks in advance for your help.

 

Ben Renquist

UCDavis