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2. Then place open end of perfusion tube in a beaker filled with
heparinized solution. Open valve (turn on the pump) and
adjust the speed you need. Fill the tube with the solution. You
never want to inject air bubbles during perfusion procedure.
Then turn off the pump for now.
3. The volume of solution should be scaled to size of animal (usu-
ally 150-200 mL for a rat of 250 g, about 30 mL for an adult
mouse for PFA and less for heparinized saline).
4. Set up surgery site with scissors, forceps and clamps.
5. The animal to be perfused is weighed and anesthetized with
either an i.p. injection of ketamine/xylazine (Xylazine (100 mg/
mL)—Ketamine (100 mg/mL) solution: mix 1:8 (v/v), inject
50-100 mL/100 g i.p. (the recommended dose is 50 mL/100 g
but it takes a too long wait before the rat gets deeply anesthe-
tized). The mix can be stored at 4°C £6 months) or induced
deeply anesthesia with isoflurane 5% for 5 min in 30% oxy-
gen/70% nitrous oxide and maintain at 3-4% during surgery.
In the first case allow 10-15 min for anesthesia to occur. Check
for nonresponse to tail pinching, or paw pinching.
6. Place the animal on the operating table under the hood with its
back down. Securely fix the animal with tape on each paw. Use
pinch-response method to determine depth of anesthesia.
Animal must be unresponsive before proceeding with the fol-
lowing steps.
7. Make incision with scissors just under the sternum. With sharp
scissors, cut through the tissue at the bottom of diaphragm to
allow access to rib cage.
8. Cut carefully the diaphragm maintaining the end of the ster-
num in forceps to avoid damaging the lungs or the heart.
9. Make two end horizontal cuts through the rib cage (on each
side) and open up the thoracic cavity.
10. Flip the sternum using safety-lock forceps (hemostats).
11. Expose the heart dissecting the thymus tissue and visualize
the aorta.
12. While holding heart steady with forceps (the heart have to still
be beating, if not the perfusion will not be successful) make a
small incision at the bottom tip of the left ventricle using
micro-scissors.
13. Insert the cannula directly into the left ventricle and go up
until you can see the cannula into the aorta.
14. Place a clamp (for example; Dietrich Bulldog Clamp, FST)
near the entry point into the heart to secure the position of the
cannula in the right place.
15. (Optional: clamp the descending aorta, which is visible along
the spinal cord, if interested exclusively in brain perfusion.)
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