[Histonet] cryostat cutting problems

[Emily Sours]

It may help to let your block equilibrate for about 20 minutes in the
cryostat.  I've found blocks do not section well if you start sectioning
right away, especially snap frozen ones.  Also, do you embed in OCT/sucrose?
A 1:1 OCT: 30% sucrose solution is much softer than just OCT (or whatever
embedding medium you are using).
What do you mean by the tissue is condensed? It's folding up? I've found
sectioning fresh unfixed tissue on a cryostat is impossible (we tried chick
embryo trunks, which may be harder to section fresh than other tissue)--it
doesn't section but get smushed to a pulp.  You need to use a sledge
microtome, unfortunately.
What thickness are you sectioning at? I've found sectioning above sixty
microns doesn't work as well on a cryostat, but again this is on fixed chick
embryo tissue.
If your cryostat is old, I would suggest you look for a new anti-roll plate
made of glass.  They work so much better.  As long as your cryostat isn't
older than about ten years, you should be able to find one that fits your
knife holder very easily.
That said, I've used a Rei-something cryostat that was 15 years old and it
sucked.  The anti-roll plate holder was too loose to do anything useful and
the adjustment of the blade holder was impossible.  Can you use a newer
cryostat in your department from another lab? Or just go back to sledge
microtome sectioning--if you know how to do it, it's the same principle
anyway.

Emily
--
Outside of a dog, a book is man's best friend. Inside of a dog it's too dark
to read.
--Groucho Marx


On Fri, Aug 13, 2010 at 4:53 PM, Entwistle, Laura <lentwistle <@t> ucsd.edu>wrote:

> I am new to using a cryostat and am having some issues with my tissue.
>
>
> 1.        Every slice seems to shatter and fragment.  When I have used a
> microtome it was because the tissue was too cold.
>
> 2.       The tissue itself was flash frozen and so was not fixed and the
> tissue did not go through the perfusion process.
>
> 3.       Each slice is condensed and doesn't lay flat.
>
> 4.       The cryostat is old.
>
> Any tips would be very helpful.
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