[Histonet] RE: to cut or not to cut mouse brains

[Connolly, Brett M]

Terry,

Here what works for us - 

Slides are kept prechilled in a -20C freezer and then kept cold in the cryostat.

Very gently press the cold slide onto the cut section so it makes contact and the section sticks to the slide.

Flip slide over and use your finger to warm the back of the slide under the section.

Starting with cold slides is a must for us. 

Brett

Brett M. Connolly, Ph.D.
Imaging Research Fellow
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_connolly <@t> merck.com
T- 215-652-2501
F- 215-993-6803





-----Original Message-----
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

Hello All,
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.
 
Signed,
Help!
Terry Kokas
 
 
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