help with cryostat cutting

Sherryl ssz1 at cornell.edu
Fri Apr 19 15:38:02 EST 1996


In article <4l0uh0$ps8 at gold.interlog.com>, brians at interlog.com (Brian
Scott) wrote:

> Hello,
> 
> I'm having difficulty cutting fixed and dissected rat hippocampus tissue 
> with a cryostat.  The slices come out with closely spaced cuts and 
> tears parallel to the blade and are too damaged to be useful.

Something you might try.  In the lab were I used to work, we would start
the brains soaking overnight in a 15% sucrose solution and then gradually
working up to 30% solution over the course of days.  This gradually pulled
all of the fluids out.  (We had problems at the two separate
concentrations because flash-freezing shredded the cell membranes.)  

As far as the shredding...when I had a similar problem, it was because the
tissues were being damaged during the freezing process (either by
dessicating or changing in some way due to ice crystal formation). To
correct the problem, I set up an ice chest lined with dry ice blocks.  I
would have crushed (powdered) dry ice in the bottom.  The stage would be
set in the ice until "cold".  I would coat the stage with fixative,
quickly position the brain, coat the entire brain with fixative and bury
the specimen in the dry ice powder.  Within a minute the brain was ready
to cut, but still remained together beautifully and did not dry out for a
while while in the cryostat.  NOTE:  We were looking mainly at the
hippothalamus & pituitary.  It was not vital that the exterior of the
brain should remain blemish free.

If you try any of this, let me know if it works.  It's been over a year so
I hope I haven't forgotten to list any steps that we used to solve the
problem.



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