[Histonet] Re: Sucrose cryosectioning

[IGOR NASONKIN]

Maria,

How do you recommend to freeze tissue after 30% sucrose and OCT imersion? Snap-freezing on dry ice or in methylbutane/fry ice bath?

I've been doing mostly paraffin sectioning until now; as i see, people have different and strong opinions on how to freeze tissue post-sucrose. Most agree that methylbutane/dry ice is a good method to snap-freeze tissue; however  i heard another, strong opinion, that this introduces some structural damage to tissue, and freezing in just dry ice is better.

However, nobody  around here immerses in OCT after sucrose, OCT is added later to hold the specimen. So, -how you freeze after sucrose-OCT. Thanks. Hope some comments will follow re. methylbutane as well.

Igor

----- Original Message -----
From: Maria Mejia <maria <@t> ski.org>
Date: Wednesday, February 22, 2006 3:53 pm
Subject: Re: [Histonet] Re: Sucrose cryosectioning

> I too have not experienced any difficulty with sectioning sucrose
> cryo material. For the past 16 years, I have sect
ioned feline & 
> primate 
> cortical
> tissue - some small and others very large  on the sliding microtome.
> 
> Both animals are perfused. The cortical tissue of interest is 
> removed & 
> tissue
> is then further fixed overnight @ 4C. Next day, tissue is rinsed in 
> PBS, 
> placed
> in 30% sucrose/0.1M PB at 4C until the tissue sinks to bottom of 
> container. I
> usually leave in this solution for another day & equally important 
> I 
> change the
> solution once a day!
> 
> After which the tissue is briefly blotted and placed into OCT 
> before 
> freezing and
> 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: maria <@t> ski.org or mbmphoto <@t> gmail.com
> Phone: (415)-621-8242
> 
> 
> 
> 
> 
> Johnson, Teri wrote:
> 
> >Weird, but I have never experience
d 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 a
dult 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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> >
> 
> 
> 
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