cryoprotection fixed brains -Reply
From: | Tony Henwood <AnthonyH@chw.edu.au> |
Nancy,
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
decreased.
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 <nmaronto@hotmail.com> 27/March/2001 08:25am
>>>
Hi,
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
adequate.
Paraffin sections of this tissue turns out very good.
They are:
1. 10%nbf/20% sucrose overnight in the refrigerator. The tissue sank to
the
bottom of the container. I embedded this tissue in OCT and froze it in the
cryostat.
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
10,20
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
a
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
appreciated.
Thanks,
Nancy Maronto
MPI Research
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