Re: Questions of Cryostat Finesse

From:Lorraine Gibbs <> (by way of Histonet)

Are these brain sections by any chance fixed in paraformaldehyde and then
cryo-protected with sucrose prior to freezing?
If so, you are not alone in having problems with bubbles and folds. The
sections will cut great but the mounting may not go as smoothly. You have
already tried just about everything.
Some people use graded sucrose solutions to get up to the final
concentration of %30. I think the results are better with fixed material if
one can get away from using just 4% paraformaldehyde. 10% formalin, or para
with gluteraldehyde, or Zambonis, may provide a firmer fixation and less
problems with the sucrose-infiltrated sections refusing to mount flat.
I generally cut sucrose-infiltrated brains a little colder (-22c to -25C).

Good Luck!

You are not alone.  If this is the fixed brain/sucrose problem, I think we
all have the problems you describe. If only the fixed specimens could mount
as well as the unfixed!

Lorraine Gibbs
Physiology & Biophysics
University Of Washington
-----Original Message-----
From: R.J. Rushmore (by way of Histonet) <>
To: <>
Date: Tuesday, December 12, 2000 4:57 PM
Subject: Questions of Cryostat Finesse

>      Hello Histonet folks,   I cut fixed frozen coronal sections of
>brain.  I cut at -20, warm on a slide warmer with nice looking  results.
>However, at a certain level, I seem to have problems.  At  this level, the
>thalamus is separated from cortical grey matter by a substantial  amount of
>white matter.  Cutting a section results in a differential  expansion - the
>grey matter on either side of the white matter expands more than  the white
>matter itself, so ruffles of tissue form.  I use subbed  coverslips to
>collect these sections, and upon collection, the ruffles of tissue  melt to
>the slide with bubbles underneath the tissue on either side of the white
>matter.  At the very least, there are notable folds.  In order to get
>around this, I have altered the following variables:  knife angle, roll bar
>angle, chamber temperature, velocity of cut and more.  I have also tried
>pressing the coverslips onto the sections, delicately attempting to paint
>the  ruffles away, warming coverslips, using slides, warming the collected
>sections  at a slower rate than on the slide warmer.  I have even lightly
>exhaled on  the section to give it more form, as a web site recommends.
>All to no  avail.     If you have any hints, or have faced this  problem
>before, could you please let me know.   Yours Sincerely,   RJ Rushmore
>Department of Anatomy and Neurobiology Boston University

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