[Histonet] freezing method AND maintain morphology ofmouse brains for LCM

From:Gayle Callis

A good friend who does lots of LCM uses dry ice cooled hexane, just sits a
beaker in dry ice and cools the hexane in a beaker. Dry ice can be added to
hexane or 2 methyl butane, but she liked cooled hexane without dry ice
better.  Dry ice, as you are doing it is actually too slow, and you can get
freezing artifact.  YOu should freeze faster than 4 minutes!  You can
either embed the tissue in OCT OR freeze the tissue without embedding and
embed it later in OCT, but still immerse in the dry ice cooled hexane.
These solvents are not good to breathe, work in a hood and NEVER store 2
methylbutane in a nonexplosive freezer, it is total disaster. 

To freeze in dry ice/2 methylbutane slurry, we cut outer edges of plastic
cryomolds (only Tissue Tek molds as other molds plastic is too thick, block
will twist out) away, leaving one end to grasp, this give a better heat
sink, or less plastic to cool, plus easier storage in 50 ml conical tubes.
Embed tissue in OCT, then hold mold so you have bottom enter dry ice
slurry, it starts to freeze, immerse and remove.  Takes only seconds and
OCT will not crack. Do overfill mold with OCT.  Freezing bottom first, then
allowing to immerse and not overfilling mold will help prevent cracks.  It
is a bit of technic involved here. 

One can also float a petri dish on liquid nitrogen, place mold inside dish
but do NOT allow liquid nitrogen to enter dish, and freeze, this is colder
than dry ice. Support petri dish with some type of holder to keep dish from
tipping over into Liq N2.  This results in the perfectly flat faced block
and you can freeze many blocks at one sitting.  


 At 02:40 PM 4/29/2004 +0200, you wrote:
>Like to hear your methods for the most rapid method of freezing tissue for
>subsequent cryosectioning, whilst maintaining best possible morphology.
>We using the PALM LCM scope to capture a small region of mouse brain, from
>fresh, rapid heamatoxylin stained ~8um cryosections. After this microarray
>analysis will be performed, so-
>**minimising RNA degradation whilst maintaining morphology are the critical
>goals here**
>Until now i have dropped the brain into TissueTek OCT medium and freezing on
>a bed of dry ice which typically takes ~4 mins or so. The morphology is
>satisfactory using this method, but working with such small amounts of
>tissue makes every second critical i feel. I just tried dropping the
>specimen (in OCT in mould) into isopentane, but a big crack formed in the
>OCT which i fear may also cause a fissure in the specimen!
>Suggestions? Experience? Greatly appreciated!
>Karl Brand k.brand@erasmusmc.nl
>Department of Genetics
>Erasmus MC
>Dr Molewaterplein 50
>3015 GE Rotterdam
>lab +31 (0)10 408 7409 fax +31 (0)10 408 9468 mob +31 (0)621 446 504
>Histonet mailing list
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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