|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:
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: Noel D. Clark [mailto:Noel.Clark@ORTHO.UAB.EDU]
Sent: Wednesday, October 01, 2003 7:45 AM
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 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