[Histonet] Re: Why dry ice isopentane slurry for snap freezing?

From:Philip Oshel

OK, I understand this. But, does ethanol or acetone at -78C really 
act fast enough to coagulate proteins in a few hours? I wouldn't've 
suspected that. Freeze substitution takes days. I don't know.
How thick a layer of OCT do you use?
But, a couple of points for the discussion:
First, you are freeze-fixing. It's reversible by thawing, but it's 
still fixation, just physical instead of chemical. Which, from what 
you are saying, is an important difference.
Second, my point with the artifact comment is that invisible 
artifacts -- i.e., below the resolution of the imaging technique used 
-- must still be considered. For instance, if you are interested in 
an enzyme that occurs inside smooth ER at some stage and moves 
outside the ER at another stage, but the freezing method, or 
subsequent handling (e.g. warming the tissue to -40C or higher, which 
is above the recrystallization point) disrupts the ER, then enzyme 
within the ER can -- almost certainly will -- move outside the ER. 
This movement may also be subresolution, but if the example enzyme is 
not accessible to the stain or Ab when within the ER, then you have a 
false positive, no matter how good the morphology.


>Simply the if, by accident, the ethanol touches any bare tissues i.e. OCT
>does not completely cover fresh  OR I decide to freeze nonembedded tissue
>ny this method (undecalcified bone) then I have a fixative in the loop.
>Isopentane does not fix the tissue, ethanol is a fixative and not always
>ideal for any immunostaining I might be doing.
>The same goes for acetone, and when I have grad students who are not as
>adept at embedding and leave tissue uncovered, then there are potential
>problems with ethanol/dry ice.  At that point, I usually go to pertir dish
>canoeing on top of liquid nitrogen - no volatile fumes other than good air
>exchange, don't want to pass out from pure nitrogen in closed spaces.
>My dry ice/isopentane method is adequate for immunostaining using a light
>microscope and some other special stains so we live with what we don't see
>even though the artifact is there. Freeze fixation is what we want to
>avoid, we prefer to fix AFTER cryosectioning.
>At 05:38 PM 10/4/2003 -0500, you wrote:
>>Gayle, Gail, & et al.,
>>A couple of things here:
>>First, why dry ice/isopentance slurry? Why not dry ice/ethanol? or
>>acetone? Yes, both are flammable, but much less so (as in not
>>explosive) than isopentane. I've done a fair amount of
>>freeze-substitution with dry ice-ethanol or acetone, and they work
>>well. And they're just as good -- or poor -- for freezing.
>>Which brings up my second point, which some of you may be tired of
>>hearing but ...
>>You do have freezing artifacts. Just because you can't see them
>>doesn't mean the artifacts aren't there and affecting your staining
>>or localizations.
>>The morphology may be perfectly good for light microscopy, and the
>>staining results may be appropriate as well. But. More rigor needs to
>>be used when working with freeze-fixation, and the exact nature of
>>the purpose of the stains or other reactions must be specified.
>>Otherwise the perfectly acceptable morphology may be badly misleading
>>as to the real nature of the stained elements.
>>There. Rant over.
>>>From one Gayle to another, a lovely setup, very stable and efficient.
>>>Curious though as to why you don't let blocks touch the dry ice?  That has
>>>never been a worry for us, as they are going from much colder liquid N2
>>>temps to approx -70 to -90C (dry ice) then into -55C freezer. When using
>>>dry ice/isopentane slurry we put newly frozen blocks on dry ice to
>>>evaporate isopentane fumes before going to -80C freezer.  I don't think the
>>>dry ice hurts anything - in fact, you would have to use it to ship frozen
>>>Do you get some kind of defect (scratch marks, etc) from putting blocks on
>>>dry ice?  We have used a solid block of dry ice to snap freeze with plastic
>>>cryomolds, OCT embedded tissue, but if the tissue is large, freezing
>>>artifact is present i.e. mouse spleen.  We do use solid dry ice on occasion
>>>to snap freeze extremely tiny NALT, or nasal associated lymphoid tissue
>>>from mouse - one of our toughest tissues to dissect out and maintain flat.
>>>We never remove the disposable plastic cryomold until just before mounting
>>>block on chuck. Obviously your method is very cost effective and you reuse
>>>the molds.
>>>Thanks for the info,
>>>Gayle Callis
>>>At 04:17 PM 10/3/2003 -0400, you wrote:
>>>>      We basically do the same thing you do.  We use a stainless steel
>>>>block sitting in the liquid nitrogen.  We fill the foam container with
>>>>liquid nitrogen covering the s.s.block, this is left for 5-10 minutes with
>>>>adding liquid nitrogen as it goes down. Once the s.s.block is cold enough,
>>>>the level of liquid nitrogen is kept below the top of the s.s. block. You
>>>>can then set your embedding boat on it. We use the Tissue Tek metal
>>>>embedding boats and embedding rings with Shandon's M-1.  The metal boat
>>>>chills and the M-1 freezes fast enough to keep away the freezing artifact.
>>>>The s.s. block is stationary and is easy to set the boat onto.  All the
>>>>surfaces of the block are flat for cutting. It is easy to do and gives
>>>>blocks and tissue.  While we are collecting tissue, we pop the block out of
>>>>the boat and put the block into a plastic bag in another foam container
>>>>dry ice to keep them frozen until we are done.  The blocks never touch the
>>>>dry ice and are transferred to a -55 freezer.
>>>>     Your way is basically the same. I don't see anything wrong with it.
>>>>    Gail Macke,HTL
>>>>    Shriners Burns Hospital--Cincinnati, Ohio
>>>>-----Original Message-----
>>>>From: Gayle Callis [mailto:gcallis@montana.edu]
>>>>Sent: Friday, October 03, 2003 10:37 AM
>>>>To: George Cole; Histonet@lists.utsouthwestern.edu
>>>>Subject: further comments on Re: [Histonet] Gayle callis' method of
>>>>freezing nerve
>>>>Correction, or rather clarification to these comments:
>>>>I do NOT immerse the bottom of the crymold DIRECTLY into pure liquid
>>>>nitrogen so this direct contact starts freezing the OCT/tissue/mold bottom
>>>>per your description.  The cryomold with OCT embedded tissue is placed in
>>>>bottom of the EMPTY platic petri dish that has been precooled by FLOATING
>>>>the dish on a layer of liquid nitrogen - basically the petri dish is
>>>>canoeing on liquid N2!  The plastic dish is not as cold as direct liquid
>>>>nitrogen contact, acts as a buffer, but still extremely cold to give
>>>>perfect artifact free freezing.  Freezing this way does not allow for any
>>>>curvature of the plastic cryomold, it stays perfectly flat. We only use
>>>>Tissue Tek cryomolds as other molds have plastic that is too thick and
>>>>conducts heat away slower than the thinner Tissue Tek plastic. One joy of
>>>>this method is once the OCT/tissue in cryomold is sitting in cold petri
>>>>dish, you can go to the next block immediatetly allowing for a large volume
>>>>of blocks/collection session.  We do not have to hold a cryomold while it
>>>>freezes. It takes a steady hand to prevent liquid nitrogen from making
>>>>contact with OCT over the top of cryomold.
>>>>When the OCT finishes freezing to top of mold with petri dish methods,
>>>>there are no indentations, just a nice layer of OCT. The petri dish method
>>>>is not as fast a freezing as you describe although it results in artifact
>>>>free tissues, and the tidge of slower freezing probably means good
>>>>interface of tissue to OCT, we never get gaps unless bubbles are not
>>>>My experience total immersion of OCT embedded tissues into liquid N2 OR
>>>>embedded in a mold results in cracked OCT, and a horrible block to section.
>>>>However, you are holding the mold to permit freezing starting at bottom,
>>>>and as said before - a steady hand helps.
>>>>Our preferred method of snap freezing is dry ice/isopentane slurry, to do a
>>>>ton of blocks at one tissue collection session.  Only when we need
>>>>perfectly flat faced blocks is the petri dish canoeing on liquid nitrogen
>>>>Gayle Callis
>>>>Research Histopathology Supervisor
>>>>Veterinary Molecular Biology
>>>>Montana State University - Bozeman
>>>>PO Box 173610
>>>>Bozeman MT 59717-3610
>>Philip Oshel
>>Supervisor, AMFSC and BBPIC microscopy facilities
>>Department of Animal Sciences
>>University of Wisconsin
>>1675 Observatory Drive
>>Madison,  WI  53706 - 1284
>>voice: (608) 263-4162
>>fax: (608) 262-5157 (dept. fax)
>>Histonet mailing list
>Gayle Callis
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)

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