Hello Sunil Thomas K,

Sucrose as a cryoprotectant is a partial dehydrant that prevents the 
formation of ice crystal artefact in frozen tissue sections -- 
especially important in the cellular 'grey matter' which freezes at a 
different rate than the highly-lipid myelinated 'white matter'. 
Also, it seems that 30% sucrose freezes at approximately the same 
speed and to the same hardness as formalin or paraformaldehyde fixed 
brain tissue.

If the tissue is frozen relatively slowly [anything less than snap 
freezing at liquid nitrogen temperatures] some kind of cryoprotection 
is crucial to prevent ice crystal artefact and to assure good 
microscopic morphology.  Ice crystal artefact, sometimes called 
freeze-thaw artefact, can be the result of no cryoprotection, too 
slow a rate of initial freezing (DON'T use the 'freeze bar' inside 
your cryoatat!)  or inadvertant warming and re-freezing of the tissue 
block during sectioning.  It will make the cerebral cortex and other 
highly cellular areas of your rat brains look 'moth eaten' under low 
power and full of large holes with distorted and shrunken cell bodies 
under higher power.  In extreme cases it can result in sections that 
are virtually useless for microscopic analysis!

The rationale for a progression through increasing concentrations of 
sucrose is consideration of simple  diffusion, as with dehydrating in 
a series of increasing concentration alcohols.  It is also an attempt 
to prevent osmotic shock to 'high water-content' cerebral cortex 
cells near the surface of the brain.  If the tissue is well fixed, 
this is a less important consideration, though the total time to 
adequate dehydration for cryoprotection will probably be about the 
same.  If the center of the tissue bloc is important, be sure to 
leave it in sucrose long enough for complete penetration ==> a crude 
rule of thumb is to leave it 'until it sinks' - thus, the specific 
gravity of the tissue and the sucrose solution are about in balance.

My 2 pennies worth for the month of April...  ;-)

-Donna

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Subject: Sucrose as a cryoprotectant
Hi, all histonetters
What is the rationale for using sucrose as a
cryoprotectant. For cryostat sectioning of rat brain
we use graded concentrations of sucrose (15%--> 20-->
30%) for cryoprotection. What is the advantage of
graded concentrations as opposed to immersing the
tissue directly in sucrose.
Hope it merits an answer.
	sunil thomas K