Clear the fix and wash bottle lines of any air bubbles. Sufficient perfusion pressure is obtained with the bottles 100-120 cm above the animal (but no more!).
Run wash solution through cannula to remove any fix from lines. Set at a slow drip.
Place the heavily anesthetized animal on its back on a rack or paraffin block over a sink. Note: A barbiturate type of anesthesia is highly preferred to gaseous types such as Fluothane, with which vessels tend to constrict resulting in poor perfusion.
Spread the forelimbs and secure each paw to the rack with a surgical towel clamp or a pin if on a paraffin block. Start running faucet water.
Make a cut along the sternum ~8 cm long, low enough to expose the sternum's end.
Grasp end of sternum with a 5" hemostat. Use sharp scissors to cut diaphragm laterally on both sides and cut toward the head across ribs and parallel to lungs.
If brain tissue is all that is desired, clamp the descending aorta (not necessary for mice). With a mosquito hemostat, reflect one lung and locate the descending aorta as it runs along the spinal column. Clamp it and allow the lung to return.
Release the hose clamp on the perfusion tube so that the fluid is just barely passing out of the cannula tip.
With left hand, use a small pair of rat tooth forceps to grasp ventral tip of the heart.
With right hand, use a #11 scalpel with blade edge out (away from heart) and pierce the left ventricle, allowing the blade to penetrate far enough to make the slit large enough for the cannula tip.
Quickly remove blade and lower heart to prevent spurts from the long incision.
With right hand, insert the cannula and direct it up through the left ventricle into the ascending aorta. Stop when the tip of the cannula is visible within the aorta.
Maintain placement of the cannula in the left hand, holding the cannula and forceps together, side by side. With the right hand free, use the rubber-tipped hemostats to clamp around the aorta, holding the tip of the cannula in place. (Clamping is difficult or not feasible in mice so the cannula/ needle must be held while perfusing.)
With left hand, release the cannula and forceps from heart and close the wash line clamp. Or you may choose to allow more wash before fix.
Fully open the fix line clamp and perfusion tube clamp.
With left hand, use the teeth of the rat tooth forceps or scissors to puncture the right auricle, allowing the escape of return circulation.
For rats, allow 200 to 250 cc of fix to flow. For mice, allow 50-100 cc of fix to flow. Consider the perfusion a dialysis process, exchanging formaldehyde and water, rather than a ‘flushing’ process.
For degeneration staining, wait at least 24 hours before extracting brain, to allow brains to fully harden and to prevent the induction of artifacts during staining.