Re: cryoprotectant
Re: cryoprotectant
--mail received 01/29/03---
Hello
Donna,
A search of
histonet on cryoprotectants netted your answer to Bret Morrow
"way" back in 2000. I work with a host of tissue, but
primarily monkey. We presently store our frozen sections in the same
glycerol/ethylene glycol solution as you. Have you since found a
solution that can handle temps as low as -40 or -70 without freezing?
It's a matter of available storage space allocation for us and not so
much an issue of tissue preservation.
Thank
you,
Donna
Donna E.
Shannon
Center for
Aging and Developmental Biology
University
of Rochester
601
Elmwood Ave. Box 645
Rochester,
New York 14642
phone:
716-273-3139
fax:
716-756-7665
Donna_Shannon@URMC.rochester.edu
Greetings from Sunny California, --Nice to hear from a HistoNet
person once again! I used to read the digest regularly, but
I've been off-line for a few years now... [cc to HistoNet is a
little Hello to the 'old gang'...]
If you have ultra-low temp storage space to spare, there is
little worry if fixed, frozen-sectioned material in cryoprotectant
freezes solid. In fact, we occasionally ship sections that
way. Just be sure that when they are thawed, it is done
gradually. For example, move samples to regular freezer temp
[-20] to thaw. Note, multiple repeated freeze-thawing cycles,
even in cryoprotected material, is generally not advisable.
Freezing temp is a function of water [buffer] content vs
'antifreeze agent' [glycerol, ethylene glycol, sucrose, etc]. I
use 50% buffer, 30% ethylene glycol, 20% glycerol--this mizture stays
liquid at -20 to -30C and freezes at -70C. You can probably
find charts for exact freezing point based on cryoprotectant
composition, or make one of your own experimentally.
If you choose to avoid freezing your samples: Before
altering the cryoprotectant composition you probably should
experiment to see if changing the buffer content results in any
alteration in preservation of tissue morphology or protein
conformation [e.g. antigens, etc] in your specific
samples. Its 'possible' that too little buffer vs
cryoprotectant might shrink and distort cells in lightly fixed tissue
or change antibody-staining or other histochemical
characteristics. Think, for example, about the differences in
staining between frozen sections and sections that have been
dehydrated and solvent-extracted for various embedding
procedures.
Another approach to the storage problem:
We buy large home-type freezers from Sears or similar for long
term storage. Do not buy "frost-free", which
regularly cycle temp almost to thawing in order to stay frost free,
and are more expensive anyway. Standard food freezer temp
of -20C is fine for months to years, and these econo-boxes are
a good alternative to expensive ultra-low storage. I set mine
at maximum cold and achieve about -30C. They can be put in
out-of-the-way places [storage room, outbuilding, basement] and left
alone for indefinite periods. We do have a monitoring system
attached to alert us of power failure in addition to occasional
on-site checks. In this long term storage mode these freezers
operate dependably for years. Uprights are more conservative of
floor space, while chest types hold temperature more
efficiently. Since there is little opening and closing, and our
floor space is limited we opt for the upright style.
If you have very valuable material, I'd suggest an electronic
temperature monitor, too--to alert you if the temp rises, even though
the electricity is on [e.g., freezer compressor failure or door left
ajar]. We used an adapted home security monitor with a
temperature probe inside the freezer until our university initiated
an integrated equipment monitoring system.
Maybe this is way more info than you wanted, but take what you
can use;-)
--
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Donna M. Simmons
HTL(ASCP),
PhC (Neuroscience Graduate Program)
Research Associate, Department of Biological Sciences
University of Southern California
Hedco Neurosciences Building
voice: 213/740-3166
3641 Watt Way - Room428
Los Angeles, CA 90089-2520
fax: 213/741-0561
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webpage: <http://www-hbp.usc.edu/people/donna.htm>
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