further comments on Re: [Histonet] Gayle callis' method of freezing nerve

From:Gayle Callis

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

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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