From: | "Charles W. Scouten, Ph.D." |
What tissue are you after? For brain, I used a modified version of
the EM protocol published by Cragg, 1980. He found a way to avoid shrinkage due to
formalin fixation.
When the fixative arrives, the sodium pump
proteins on the outside of the cell are the first to be fixed. Sodium rushes into the cell down the
concentration gradient, and water rushes in to maintain tonicity. The cell swells 30%, and fixation occurs
with crosslinking to proteins in neighboring cells. Later, when equilibrium is restored and the
cells shrink back, they drag their neighbors back with them. The result is 20% gross shrinkage of the
brain, and loss of extracellular space.
Cragg reasoned that if you could replace the extracellular fluid with isotonic fluid without sodium, the
shrinkage would not occur at all.
Isotonic sucrose (close to 5%) fills the bill, but will not replace extracellular fluid because of the blood brain
barrier. This can be overcome if
the sucrose is pumped through at 300 mm Hg to overcome the blood brain barrier
without breaking blood vessels.
We offer a suitable apparatus to implement
this plan, see the link below, and a protocol. It has the salutary effect of completely
and thoroughly washing out the red blood cells. You use sucrose for the prewash instead of saline, very briefly because of the high
pressure and flow rate, then get the fixative to the cells
more rapidly than traditional methods.
Gravity flow is too weak, 150 mm Hg
translates to 6.7 ft of water. In
your lab space, can the water reservoir be 6.7 ft above the animal on the sink,
without raising the ceiling? Or to
implement the Cragg perfusion, you would need 13.4
ft. from animal to sucrose. Peristaltic pumps control flow
rate, which does not correct for vascular resistance of the animal, and the correct
flow rate is not obvious. Try the
Perfusion One:
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: Noel D. Clark
[mailto:Noel.Clark@ORTHO.UAB.EDU]
Sent: Wednesday, October 01, 2003
7:45 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Perfusion
techniques in mice/rats
Was hoping to get input on the
various perfusion techniques used by other research labs for rodents. As
always, any help is greatly appreciated. See you in Louisville!
Thanks in advance,
Noel
Noel Clark, M.A., HTL (ASCP)
1919 7th Ave South
Orthopaedic Research Laboratory
Center for Metabolic Bone Disease
University of Alabama at Birmingham
Birmingham, Alabama 35294