cryoprotection fixed brains -Reply

From:Tony Henwood <>

Why do you need to do the OCT or sucrose infiltration under
refrigeration. Since the brain is well fixed in formalin, I would try the
infiltration at room temperature. The chance of holes should be greatly
Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

>>> Nancy Maronto <> 27/March/2001 08:25am
I need your help.  I have a project that is using 10% neutral buffered 
formalin perfused fixed brains that need to be sectioned with a crostat.  I

have tried three methods that were suggested and found that all three 
created vacuoles in the tissue section.  The cryoprotection is not
Paraffin sections of this tissue turns out very good.
They are:
1. 10%nbf/20% sucrose overnight in the refrigerator. The tissue sank to
bottom of the container.  I embedded this tissue in OCT and froze it in the 
2.  Fixed tissue infiltrated in 50% OCT overnight in the refrigerator. Next 
day embedded tissue in 100% OCT and froze in cryostat and cut
sections. This 
works very well on other tissues such as livers.
3.  Three concentrations of sucrose 1 day each under refrigeration
and30%.  Each day tissue was at bottom of container before changing 
solution.  This tissue was embedded in OCT, frozen and cut.

Method 3 turned out the best, however, not good enough. We have only
cryostat to use to cut this tissue.  Is it possible to cryoprotect this NBF 
fixed tissue to get good frozen sections? Any help would be

Nancy Maronto
MPI Research
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