RE: Freezing muscle; OCT etc; cryoprotection

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From:"J. A. Kiernan" <>
To:jim <>
Date:Mon, 5 Jul 1999 23:48:42 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Tue, 6 Jul 1999, jim wrote much, including:

> Now to your point: A cryoprotectant which only surrounds and not infiltrates
> the specimen can only be useful for the second method, since such a medium
> contributes to the bulk of the specimen and does not lower the freezing point
> of the specimen itself.

   Obviously this is true. I don't think even the makers of OCT
   compound and other such goo would claim a cryoprotective action.
   These materials serve to glue the frozen specimen to the
   cryostat chuck or bit of cork, and also provide some protection
   against sublimation of the ice (freeze drying) in stored frozen
   specimens. Gelatin (approx 2%) has a similar consistency and does 
   the same job more cheaply. 

> To be effective during vitrification the cryoprotectant 
> must infiltrate the specimen.

   It must indeed.
   A.G.E.Pearse ("Histochemistry" Vol 1) makes the point that
   most of the cryoprotectives used with tissues - sucrose being
   the most popular one - penetrate only the extracellular
   spaces. He says that the insides of cells are quite well
   cryoprotected already by their own dissolved hydrophilic
   macromolecules. A recent publication shows that for
   vitrification with sucrose the concentration must be at
   least 60% (Lepault,J., Bigot,D., Studer,D. and Erk,I. 1997.
   J. Microsc. (Oxford) 187, 158-166). It's unusual to go
   above 15 or 30% for cryoprotecting fixed specimens for
   subsequent LM, and these lower concentrations seem to do
   quite a good job for CNS and muscle prior to cutting
   thick (30-100 um) sections. The ice crystal holes must be 
   too small to see.  

   For many purposes muscle must be unfixed ant cut at 5-10 um.
   Muscle cells can't have enough of their own cryoprotective 
   proteins, because you certainly get ice crystal holes within
   the fibres: either biggish central holes or multiple tiny
   holes. The latter are rather regularly spaced among
   the myofibrils and not offensive for jobs like staining
   for ATPase or myosin isoforms. This description is of what 
   I've seen. Ian Montgomery might well provide a more
   accurate interpretation, but I'm sure he'll agree that the
   big central holes are seen when something goes wrong and
   freezing happens too slowly. 

   Does anyone know why DMSO (dimethylsulphoxide) isn't used
   often for cryoprotecting unfixed specimens? Pearse cites
   evidence of it being better than glycerol or sucrose, and
   at lower concentrations, and I'm told it's used regularly
   to protect cultured cell lines from death by freezing.
   That's my 2d/2p/2c-worth.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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