Re: OCT: What's in it? Is it good? Etc (Rather long)
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|From:||"J. A. Kiernan" <email@example.com> (by way of histonet)|
On Tue, 11 Jan 2000, Linda Prentice wrote:
> Hi I've just had a discussion with a researcher who claims that OCT
> contains sucrose. I've always believed that OCT had no cryoprotecting
> quality and that it was simply an embedding medium. ?????
There is a short answer to your question, Linda, and you
know it already.
OCT certainly doesn't act as a cryoprotectant, even if it does
contain sucrose, because it doesn't infiltrate the specimen. As
you say, it just surrounds it and enters crevices, providing some
physical support. It may also retard evaporative loss of water
(freezer burn) in the cold air of the cryostat, if it completely
encloses the specimen. Water would also do this. Cryoprotectants
(sucrose, glycerol, DMSO etc) have to soak into every part of
the tissue, replacing a significant proportion of its water.
Unfortunately the identity of OCT isn't stated on the
squeezy-bottle, and the same is true of other brand-name
products that do the same job. A reasonable guess might
be that they contain water and polyethylene glycols of
quite high molecular weight. Gelatin (about 2% in water)
seems to work about as well. Some HistoNet discussion in
the past indicated that the initials stood for Optimum
Cutting Temperature. This makes no sense in the context
of cryostat sectioning, but it may do so if you are using
an old-fashioned open-air freezing microtome (as do many
of us who work with thickish sections of CNS, muscle or
skin for research purposes).
On a freezing microtome the sections thaw as they are being
cut and are then collected off the blade with a paintbrush,
though I've seen at least two operators who used a clean
finger, claiming that it could be more delicately controlled.
(Yes. I have compared brush with digit, and I preferred the
former; camel hairs don't dull the cutting edge.) A specimen
must be only just below freezing for getting good sections. If
it's too cold they shatter whn cut, and if the block's not solid
it won't cut at all. The better and more expensive freezing
microtomes let you control the temperature of the chuck, either
by circulating refrigerated alcohol inside it or by means of
a Peltier effect thermoelectric device. With cheaper ones (like
mine) the specimen is quickly frozen with carbon dioxide and
the sections must be cut just before the block begins to thaw.
To return to OCT: This material has a higher melting/freezing
point than water, and this gives it a slight advantage as a
supporting medium when cutting sections on a freezing microtome.
The OCT stays more or less solid even when the enclosed specimen
is too soft to cut. The support prevents the specimen from
being knocked off the chuck by the knife. The operator can
re-freeze with another blast of CO2. Freezing with CO2 on an
old freezing microtome is remarkably fast and efficient, and
ice crystal holes are rarely seen, even if you don't cryoprotect
the specimen with sucrose.
There is a down-side to OCT, gelatin and other polymeric support
substances. They do not go away, and you end up with gooey
material in the grooves of your microtome chuck, all over your
knife, around every section (cryostat) or mixed up with the
free-floating sections (cryostat or freezing microtome). A simple
alternative is to support the specimen with distilled water. This
leaves no residues and cuts easily on a freezing microtome or
cryostat. The only disadvantege of water, IMHO, is that on a
carbon dioxide microtome you are more likely to knock the block
off than you are if you use OCT or an equivalent material.
HistoNetters of the world, reply! If you have compared various
commercial products with one another or with something cheaper
like water or gelatin, tell us all what tissues you work with
and what you think of the various materials that may make it
easier to cut frozen sections.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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