[Histonet] storage of cryosections

[Philip Oshel]

I'm afraid I have to disagree with a portion of John's email: ice 
crystal damage can happen in storage.
Ice crystal growth and refreezing occurs at temperatures above about 
-60 to -40  deg C, depending on the specifics of the tissue, local 
environment, and all the rest of the annoying biological realities we 
deal with.
If tissues are frozen rapidly enough to prevent crystal formation, or 
to form only tiny crystals, the tissues/cells will experience crystal 
growth if they are warmed above the recrystallization temperature. 
Much of the damage is actually caused by dehydration as the growing 
ice crystals pull free water out of cells and intercellular spaces, 
but as crystal growth continues, it can form holes.
The preventative is to store samples in -80 deg C freezers and to 
warm them too quickly for recrystallizaton and crystal growth to 
occur.
But, there is an even neater trick: store the samples under vacuum in 
a -80 freezer with *lots* of desiccant (Lots -- no, more ... pile it 
in the chamber. More!*). Something with a high water capacity: best 
is 3-4 Angstrom (4 is better than 3, 5 is no good) molecular sieve, 
next best is silica gel. Leave the samples like this for a few days 
(hours to weeks, depending on sample size and number -- it's 
empirical, try it and see), and they will be nicely freeze-dried by 
vacuum sublimation. They can then be stored at any temperature with 
desiccant with no worries about ice crystals, or much of anything.
On exposure to air, the samples will immediately start rehydrating, 
so they *must* be at room temperature before removing them from the 
desiccant chamber, and processed right away.
Caveat: I don't think this would adversely affect antigen epitopes, 
but this should be tested.

Phil
* If you think you have enough, you don't.

>Freezing artefacts (ice crystal holes) form while
>the tissue is being frozen, not during storage of
>sections. Once the holes are there, because of
>too-slow freezing, nothing can get rid of them.
>
>Drierite (or a similar desiccant) ensures that
>the stored sections are in a dry atmosphere.
>You should let the box warm to room temperature
>before you open it; otherwise water from the air
>will condense on the cold sections. If they are of
>unfixed tissue this will cause some damage, which may
>or may not matter depending on what you intend to
>do with the slides.
>
>John Kiernan
>London, Canada.
>------------------------------------------------
>Birthe Schnegelsberg wrote:
>>
>>  HI.
>>  Do I actually have to add Drierite absorbent into the boxes with the cryo
>>  sections in the -20C freezer to avoid freezing artefacts?
>>  Thanks, birthe
>>
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-- 
Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)