RE: [Histonet] Lovely set up for snap freezing

From:Philip Oshel

Interesting. How can the isopentane be colder than the dry ice? The 
ethanol or acetone is at dry ice temperature, once equilibrated.

>Take temperatures of your slurry, you will find that the temperature 
>with isopentane is 20-30 degrees colder.
>	-----Original Message-----
>	From: Philip Oshel []
>	Sent: Sat 10/4/2003 3:38 PM
>	To:
>	Cc:
>	Subject: Re: [Histonet] Lovely set up for snap freezing
>	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 []
>	>>Sent: Friday, October 03, 2003 10:37 AM
>	>>To: George Cole;
>	>>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
>	>>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.
>	>>>
>	>>
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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
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