The saline prewash would be just lost time.  It accomplishes nothing that will not be accomplished by the sucrose, which is equally effective at washing out the blood (more effective given the high pressure). Start with the sucrose.  Time is of the essence, tissue deterioration begins immediately upon anoxia.  Switch to fixative when the animal's muscles cease to move.

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


-----Original Message-----
From: Nancy.Walker@sanofi-synthelabo.com [mailto:Nancy.Walker@sanofi-synthelabo.com] 
Sent: Friday, October 03, 2003 8:12 AM
To: Charles W. Scouten, Ph.D.; Histonet@lists.utsouthwestern.edu
Subject: shrinkage and Perfusion One system


Hello,

From the website mentioned in C. Scouten's mail I recuperated this info:

The shrinkage occurs when a prewash with physiological saline is followed
by fixative. The first action of fixative on the cell membrane is to shut
off the sodium pump proteins. Sodium rushes into the cell, followed by
water to maintain tonicity, and cells swell and expand. Membrane proteins
are fixed and crosslinked to neighboring cells in the swollen position.
Later, the cells stabilize and contract, and pull other cells with them.
The result is gross shrinkage, local distortion and torn membranes with
lost cellular contents from some cells.

Tissue Shrinkage can be completely prevented if the extracellular fluid
with ions is replaced by a nonionic isotonic solution that cannot enter the
cells. This can be accomplished by prewash with 5% (isotonic) sucrose in
distilled water. Start the prewash at low pressure, and pump up to 300 mmHg
to break the blood brain barrier and wash the extracellular fluid (and red
blood cells). Switch shortly thereafter to fixative. The perfusion takes
the same time as the traditional procedure, and accomplishes the favorable
results bulleted above.

So if I'd like to try out the perfusion techniques that are made simple by
the Perfusion one system, what would you suggest:   first do a
physiological saline solution, followed by a prewash with 5% sucrose and
then PFA, or go directly to 5% sucrose then PFA?

thanks for your advice,

Nancy




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