Re: [Histonet] Frozen embedding of lungs
Why not inflate with air (Pasteur pipette in
trachea) and then plunge into isopentane (cooled
by liquid nitrogen) or acetone (with dry ice ion
it). Neither is ideal, but both freezing methods
protect quite well against ice crystal damage; the
crystal holes are much smaller than cells.
Coating a specimen with goop before freezing
ensures that the cells of the specimen are the
last and most slowly frozen things in the blob.
That makes for maximum damage by ice crystal
formation. With slow freezing most of the the ice
crystal holes are about the same size as a cell,
and the tissue architecture is 100% wrecked.
Unfixed objects must be frozen alost instantly. A
blob of hydrophilic polymer can be added
afterwards to protect against freeze-drying of
stored specimen. OCT (whatever it is; ?polyvinyl
alcohol) is a physical support for specimens being
sectioned with a cryostat or an olde-worlde
freezing microtome. It does not infiltrate the
tissue in the way paraffin wax enters every
dehydrated crevice. Most of the functions of OCT
are duplicated by other hydrohilic polymers such
as gelatin or agar.
The abbreviation OCT is for Optimum Cutting
Temperature. That has nothing to do with
protection against freezing artifacts. It relates
to the hardness of the polymer at some temperature
that was, many years ago, considered optimal for
cutting frozen sections.
Kim O'Sullivan wrote:
> Hi all,
> Does any one out there have a successful method for inflating mouse lungs to freeze. We currently freeze them unfixed in OCT, but of course the morphology is shocking as the lungs have collapsed. Is there a method that preserves the morphololgy without collapsing or imploding the lungs.
> Any advice will be appreciated
> Kim O'Sullivan
> Centre for Inflammatory Diseases
> Department of Medicine
> Monash University
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