RE: Freezing muscle; OCT etc; cryoprotection
<< Previous Message | Next Message >>
From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | jim <jim@proscitech.com.au> |
Reply-To: | |
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
<< Previous Message | Next Message >>