Re: [Histonet] Re: Sucrose cryosectioning
I too have not experienced any difficulty with sectioning sucrose
cryo material. For the past 16 years, I have sectioned feline & primate
tissue - some small and others very large on the sliding microtome.
Both animals are perfused. The cortical tissue of interest is removed &
is then further fixed overnight @ 4C. Next day, tissue is rinsed in PBS,
in 30% sucrose/0.1M PB at 4C until the tissue sinks to bottom of
usually leave in this solution for another day & equally important I
solution once a day!
After which the tissue is briefly blotted and placed into OCT before
sectioned. Although, I have not left my sample sit in OCT for up to an
hour - I do
leave the sample in 4 degree OCT for at least 15-20 minutes.
Hope this helps.
Maria Bartola Mejia
Smith-Kettlewell Eye Research Institute
San Francisco, CA 94115
Email: email@example.com or firstname.lastname@example.org
Johnson, Teri wrote:
>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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