[Histonet] Re: Sucrose cryosectioning

From:"Johnson, Teri"

Weird, but I have never experienced difficulty with sectioning sucrose
cryopreserved material.  I've heard Gayle Callis mention how awful it
is, and now others say they have problems with it. (I'm almost afraid to
speak up that I may jinx myself!)

 I agree with Andi, it's a good idea to allow the sample to sit in OCT
for a bit before freezing, but on some of our chick and mouse embryos,
we have embedded in OCT and then quickly frozen with good results.

We have sectioned mouse testis, femur, eyes, and pancreas all treated
this way and never had an ooey gooey mess, even at -20 to -25 C.  For
the larger adult samples, I think you probably do need to have the
cryostat colder than you would for unfixed frozen samples.

We formalin fix 2 hours for small samples, to overnight for large
samples, then rinse in PBS, place into 15% sucrose until they sink, then
place into 30% sucrose until they sink. Remove as much of the sucrose as
you can (briefly blot adult samples) before placing into OCT, and allow
to sit up to an hour before freezing (cover, so the OCT doesn't harden!)

Pancreas has turned out to be the worst in terms of morphology for us.

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133

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