Re: [Histonet] Lovely set up for snap freezing

From:Philip Oshel

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.

Phil

>Gail,
>
>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
>blocks.
>
>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:
>>Gayle,
>>     We basically do the same thing you do.  We use a stainless steel square
>>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 great
>>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 with
>>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:
>>
>>George,
>>
>>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
>>removed.
>>
>>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
>>used.
>>
>>Gayle Callis
>>MT,HT,HTL(ASCP)
>>Research Histopathology Supervisor
>>Veterinary Molecular Biology
>>Montana State University - Bozeman
>>PO Box 173610
>>Bozeman MT 59717-3610
>>
>>
>>
>>At 04:56 PM 10/2/2003 -0700, you wrote:
>>>         Histotechs, Gayle Callis’ method of freezing nerves is
>>>illustrated in the DVD’s in the Muscle and Nerve packet I’ve
>>>been send around.  It shows a nerve in a flat plastic mold covered with OCT
>>>It stresses the fact that the liquid nitrogen must touch the bottom of the
>>>mold only. It must not get into the top of the mold. Freezing causes the
>>>OCT to contract.  Freezing from the bottom like Gayle says ands the DVD
>>>shows, keeps the contraction to only a small dent on the surface of the
>>>frozen OCT.  No problem.  This is shown in the DVD. It’s easily
>>>filled. If the liquid nitrogen gets into the top of the of the mold, the
>>>contraction will take place around the nerve making sectioning difficult
>>>indeed.  Athough DVD One band 9 shows how to patch gaps like these,
>>>there’s no reason to do so if you freeze the tissue carefully.
>>>georgecole@ev1.net
>>
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>
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-- 
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)

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