Re: Newbie has a problem (Gelatin embedding)
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Tue, 8 May 2001, Bonnie Wayne wrote:
> I am having some difficulties with my histology work. I have rat cerebellum
> and brainstem embedded in gelatin, sliced, and stained with cresyl violet.
> 1) The gelatin likes to curl. The brain section (30 um) sticks
> wonderfully but the excess gelatin loves to lift off of the slide.
> ... Any suggestions on how to get the gelatin to stick?
There is an ancient method of true gelatin embedding that was used
150 years ago, before the introduction of paraffin, formaldehyde
or freezing microtomes. It allowed cutting of pretty thin sections,
we're told, and gave good cytological preservation, but nobody
would use such a time-consuming and complicated method these days.
The way it's usually used, gelatin does not infiltrate an object;
it surrounds it and enters the larger crevices. Its cutting and
properties and stength are enhanced by formaldehyde fixation of
the "embedded" and trimmed block. After 12-24 in the fixative
the gelatin is cross-linked enough to be insoluble. It also
won't melt when warmed, and you have nice square frozen sections
that are more easily manipulated than brain-shaped ones. You didn't
say if your gelatin blocks were fixed. You also didn't say how
long the brain was fixed for, or if a cryoprotectant (it's usually
surose) was used, either before or after putting in gelatin. It
makes sense to do the cryoprotection last, in the hope of getting
the most even consistency of tissue-plus-gelatin.
Section adhesion is also affected by the way you mount the sections
and by any pre-treatments that you apply to the slides (cleaning,
adhesives etc). For obvious reasons mounting from water is preferable
to mounting from a solution containing dissolved salts that may end
up as insoluble crystals under the sections.
Just a few ideas; there may well be more than one cause for the
difficulties you describe.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
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