[Histonet] storage of cryosections

[Charles Scouten]

I agree that initial freezing is most important, but that freezing artifact can develop in storage because the ice will reform and crystalize.  See the following link for a thorough discussion.

http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freezing%20Artifact.pdf

Drying agents are thus a good idea.

 


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 
http://www.myneurolab.com 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Philip Oshel
Sent: Tuesday, December 14, 2004 9:01 AM
To: jkiernan <@t> uwo.ca
Cc: Histonet <@t> Pathology.swmed.edu
Subject: Re: [Histonet] storage of cryosections

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)

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