Yes, definitely make sure to do the 20% sucrose overnight, even try 30%, or
grades going from 10%-20%-30%, switching to the next higher when the brain
becomes saturated enough to fall to the bottom of the container. We did
this in a previous lab with rat brains and it worked well.
Merced
--On Friday, March 06, 2009 11:52 AM +0900 shymaa shawadfy
<sshaw...@med.kobe-u.ac.jp> wrote:
Dear all
I am trying to use vibratome 50 µm thick sections for immunofluorescence
using Postnatal day 0 brains. The problem is that brains are very soft and
are usually destroyed upon handling and the agarose is separated form the
brain.
My used protocol was: perfusion with 4 % PFA for 3 min, followed by
several hours to overnight post-fixation. Then embedding brains in 2 %
low melting agarose and cutting the block on vibratome using low speed.
I am thinking to add an overnight 20 % sucrose incubation step following
the post-fixation step. Then embed in agarose and continue the normal
protocol. May be sucrose will increase the elasticity of the tissue.
So what do you think ?
Thanks a lot
shymaa
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Merced M Leiker
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