[Histonet] Lovely set up for snap freezing
Philip Oshel
peoshel <@t> wisc.edu
Sun Oct 5 08:31:21 CDT 2003
Interesting. How can the isopentane be colder than the dry ice? The
ethanol or acetone is at dry ice temperature, once equilibrated.
Phil
>Take temperatures of your slurry, you will find that the temperature
>with isopentane is 20-30 degrees colder.
>
> -----Original Message-----
> From: Philip Oshel [mailto:peoshel <@t> wisc.edu]
> Sent: Sat 10/4/2003 3:38 PM
> To: Histonet <@t> Pathology.swmed.edu
> 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 [mailto:gcallis <@t> montana.edu]
> >>Sent: Friday, October 03, 2003 10:37 AM
> >>To: George Cole; Histonet <@t> 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 <@t> ev1.net
> >>
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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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