[Histonet] RE: to cut or not to cut mouse brains
hfedor <@t> jhmi.edu
Thu Nov 17 13:51:57 CST 2011
These are very difficult. The colder the better. (-27). keep the slides in the cryostat. Cut the section. Place section on a cold slide, and keep the slide in the croystat during the following procedure.
Hold slide in left hand and brush in right. Place finger under one edge of the section and it will start to thaw, anchoring it to the slide. Gently brush out folds as you continue to advance the thaw line with your fingers under the slide. It takes a bit of practice, and not really perfect. But it works.
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of McLaughlin, Terry
Sent: Thursday, November 17, 2011 2:25 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] to cut or not to cut mouse brains
I am having trouble cutting frozen mouse brains and was wondering if
someone can offer some help. The mouse was perfused in 4% PFA , the
brain removed and kept in PFA for 22 hrs. We placed it in 30% sucrose
for 20 hrs, until the brain sank in this solution. It was frozen back by
using a culture plate floating in liquid nitrogen with OCT .
The problem I am having is many folds, and air bubbles under the tissue.
What I have done so far:
Cut at temp of -25, then tried - 22 and -17.
Cut at 10um, then tried 16-25um.
Tried making the knife colder by adding dry ice to it for a few seconds.
Added dry ice to sample for a couple seconds.
The sample cuts fine, it appears to happen when picking up on a slide. I
tried picking up by starting at one side or end and letting tissue
spread onto slide. Did not work.
Also tried gently laying slide on top of sample and removing
gently-still folds and air bubbles. Any help would be greatly
appreciated as we have 6 more to do.
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