Not "rapid" freezing!
|From:||"J. A. Kiernan" (by way of histonet)|
The following passage occurred in part of a huge multiply
quoted Histonet email under a "Re - Daily Digest" subject,
which in some half-asleep state I read instead of deleted.
(If only digesters could be automatically shut out, along
with the unscribers!) There is a need to respond, because
internet search engines pick up Histonet messages, and there
is no shortage of gullible websters.
> ... whole mouse brain ... will freeze completely in
> about 3-5 minutes.
Let us hope that nobody will take this utterance seriously.
It was part of a lengthy account of a tissue freezing method
that could not possibly yield acceptable results for
microscopy. Even with conventional cryoprotection an object
that takes 3 minutes to freeze will be full of ice crystal
holes. Each hole is about the size of a large cell, and is
separated from neighbouring holes by damaged (compressed)
bits of tissue 2-10 times the width of a big neuron.
A specimen the size of a mouse's brain (least dimension
about 7 mm) can be frozen (after adequate fixation, followed
by cryoprotection) by dunking in isopentane or propane at
liquid nitrogen temperature, or by an acetone-dry ice
mixture. If you want thick sections, use an old freezing
microtome, with a CO2 siphon.
For microscopy, you must freeze in seconds or fractions
thereof; even one minute is too long.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
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