RE: [Histonet] Re: Cryosections from formalin fixed tissues

From:"Monfils, Paul"

Sucrose doesn't "preserve" the tissue in the same sense that fixatives do.
Rather it protects the structural integrity of the tissue which would
otherwise be disrupted by the formation of ice crystals. A concentrated
sucrose solution freezes as a microscopically homogeneous matrix, without
the formation of large crystals as seen during the freezing of most aqueous
solutions. This helps in two ways. First, ice crystals as they form
perforate cell membranes and other delicate structures, and also tend to
"push aside" the surrounding tissue, leaving spaces once the ice eventually
melts.  Secondly, ice does not slice smoothly, so the more ice is present in
a tissue, the more difficult it will be to produce a smooth slice of that
tissue. The sucrose matrix slices much more smoothly than ice does. Tissue
that has been in formalin or another aqueous fixative (or which has been
washed in water or buffer) naturally contains a large amount of water, and
therefore freezes with a high degree of ice formation.

I have occasionally kept tissues in sucrose solution for several weeks
without adverse effects, though I don't do so routinely. About the only
possible problem that might occur would be the growth of microorganisms in
the solution; but even that is unlikely due to the high osmotic pressure of
such a solution (we don't have to refrigerate corn syrup or honey, for
example).  Though I have not tested Oil Red O staining after long sucrose
exposure, it seems unlikely that there would be an adverse effect since fat
is immiscible with aqueous solutions, and I can't think of any actual
reaction that might occur between the lipids and the sucrose.

Sections can be returned to formalin after cryosectioning with sucrose, and
if the tissue was well infiltrated with the sucrose solution and properly
frozen, there is typically minimal if any detectable freeze artifact. We did
this most recently with some whole rat brains which had been
sucrose-infiltrated and frozen, which we subsequently needed to embed in
paraffin. If the tissue is frozen in a tissue freezing medium, I just place
the whole frozen block into the formalin, and allow it to thaw there at room
temparature.  I tried thawing them in the refrigerator, but found that the
tissue embedding medium was reluctant to dissolve into the formalin at that
temperature.  I leave them in formalin overnight, both to allow the
embedding medium to dissolve, and to allow the sucrose solution to diffuse
out of the tissue. Then  I transfer them to fresh formalin.

> ----------
> From: on behalf of
> kotresh mattur
> Sent: 	Saturday, August 20, 2005 5:41 AM
> To:
> Subject: 	[Histonet] Re: Cryosections from formalin fixed tissues
>  Hi dear all,
>  We r trying to get cryosections from 10% NBF fixed tissues for Oil Red
> 'O' staining. 
>  First we   kept the fixed tissues for washing under tap water for
> overnight and then tried cryosectioning where we didnot get the quality
> sections
>  Later we tried by keeping the tissues under 20% sucrose solution under
> refrigeration overnight(as mentioned by Gayle callis )and then we got
> better quality sections compared to the above said method. 
> still we have few querries, can anybody give us ideal procedure for the
> same with your practical experiences? 
> 1. How does sucrose acts in preserving the tissues? i have read that it
> acts as cryoprotectant but i want to know how exactly it helps 
> 2.If we r keeping the 10% NBF fixed tissues in 20% sucrose solution then
> for how many days the tissues can be kept in the same solution under ref.?
> If so does it affect or come in the way of Oil Red 'O' staining and
> evaluation? 
> 3.After taking cryosetions if i want to preserve the same tissues whether
> they can be placed back to  10% NBF ?
> Thanks in advance 
> Dr.K.Y.Mathur
> Dr.K.Y.Mathur
> Junior Scientist
> Dept.of Preclincal Safety Evaluation
> Discovery Research
> Dr.Reddy's Laboratory Ltd.
> Bollaram Road, Miyapur,
> Ph No: 
> 040-23045439-exn 424,421,422,423
> Cell :09391293129
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