Re: cryoprotection fixed brains etc

From:"J. A. Kiernan" <>

Short comments on Nancy M's three methods are followed 
by a few queries, suggestions and remarks (relating to 
fixation and how to spend money).

On Mon, 26 Mar 2001, Nancy Maronto wrote:
> have tried three methods that were suggested and found that all three 
> created vacuoles in the tissue section. The cryoprotection is not adequate. 

That is spot-on. The "vacuoles" are holes made by ice crystals.
Perfection is almost impossible to attain, except in very thin
layers of cells, but it is possible to have holes that are either
too small to see with LM or (if visible) small enough not to
disturb the structure too much.

> 1. 10%nbf/20% sucrose overnight in the refrigerator. The tissue sank to the 
> bottom of the container.  I embedded this tissue in OCT and froze it in the 
> cryostat.

20% sucrose isn't strong enough, but it's better than nothing. The
counsel of perfection (from those who have done EM on frozen solutions)
is 60% sucrose. Most people seem to settle for 30%, which isn't so

Freezing in a cryostat cabinet isn't fast enough. 

> 2.  Fixed tissue infiltrated in 50% OCT overnight in the refrigerator. 
> Next day embedded tissue in 100% OCT and froze in cryostat 

OCT and other such gooey substances are for providing physical
support while cutting, and can also reduce drying-out (= freeze-
drying, or freezer burn) while a specimen is in very cold air.
These water-miscible polymers do not infiltrate the tissue in the
way as melted wax, resin monomers, or nitrocellulose, and they
cannot cryoprotect.  A cryoprotectant has to have small molecules
that go into all the cells and extracellular spaces - everywhere
where there's water.
> 3.  Three concentrations of sucrose 1 day each under refrigeration 10,20 
> and 30%. Each day tissue was at bottom of container before changing 
> solution.  This tissue was embedded in OCT, frozen and cut.

> Method 3 turned out the best, ... 

So it should. 
>                 ... however, not good enough.

Faster freezing will solve your problem, and I'm sure you
will get plenty of advice about this, so won't say anything,
BUT there is one other consideration.

> ... Is it possible to cryoprotect this NBF fixed tissue ...

Are the brains actually fixed?  Formaldehyde (especially when
buffered to a near-neutral pH) works more slowly than any other
fixative, even when it's delivered to tissues almost instantly
by vascular perfusion. Unless the requirements of an investigation
necessitate minimal fixation by formaldehyde, the specimen
should be immersed, after perfusion, at least overnight. It's
OK to include some sucrose in the fixative. Published mixtures
include 15% and 30%. I can send refs if you ask.

In the Histonet archives ( you will find 
plenty of contributions that support all these statements 
(and NO, they aren't all from me).

There is also plenty of printed literature, which is more reliable 
than the Internet because much of it has been peer-reviewed. If a 
good library isn't available, the best investment any lab can 
possibly make is in books. The most comprehensive and authoritative 
of these, in the histochemical field, is A.G.E.Pearse's
"Histochemistry," 3rd edn in 3 volumes, 1980-1991 published by 
Churchill-Livingstone. It costs about $300 for the whole set, with 
hard covers and glossy paper.  Compare this with the prices of a 
few ml of an antibody that won't work, or a Justin Case chest X-ray
or CT scan, or having a few gallons of wholesome but slightly used 
ethyl alcohol taken somewhere and ignited with the expenditure of
one match. 

'nuff said!

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


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