Questions of Cryostat Finesse
From: | "R.J. Rushmore" <rushmore@bu.edu> (by way of Histonet) |
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 rushmore@bu.edu
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