Re: cryoprotectant

From:Donna Simmons

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 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

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;-)

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
webpage: <>

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