[Histonet] mouse brains- sucrose cryoprotection- sectioning

[Charles W. Scouten, Ph.D.]

This is a well known effect, mentioned in the atlas of Konig and Klipple (precursor to Paxinos).  You didn't say, are you perfusing the brains, or just extracting them and dropping them in PFA.  Definitely, you should perfuse for better quality, to get fixative everywhere fast.  

Either way, you will get shrinkage and distortion unless you perfuse with a sucrose prewash at 300 mmHg, followed by the fixative.  An apparatus is available, a protocol is posted, and the rational is given at the following link.

http://www.myneurolab.com/myneurolab/mnl_products_detail.asp?idproduct=471001&catdesc=Histology+Equipment&CatThreeID=674&CatOneID=4&subcatdesc=Sacrifice+Equipment&idsubcategory=21


Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 
www.myneurolab.com 


-----Original Message-----
From: Yogeshwar Kalkonde [mailto:kalkonde <@t> uthscsa.edu] 
Sent: Monday, October 27, 2003 4:29 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] mouse brains- sucrose cryoprotection- sectioning

Hi Histonetters-
i am trying do Nissl staining on mice brain sections. Mice brains were fixed 
in 4%PFA and cryoprotected by overnight treatment with 30% sucrose. Brains 
were then embedded in OCT and frozen.
while trying to section the the tissues in a cryostat (@8 microns thickness) 
the sections shrink, distorting the tissue architecture. 
any clue what might be going wrong?
thanks in advance...
yogesh

Yogeshwar V. Kalkonde, MD.
Post Doctoral Fellow,
Department of Medicine (Infectious Diseases)
University Of Texas Health Sciences Center at San Antonio, 
7703 Floyd Curl Drive, 5.084R, Mail Code 7870,
San Antonio, TX 78229-3900
Ph: 210-567-0385 (lab)


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