Intra-cardiac mouse perfusion

Anesthetize with a lethal dose of pentobarbital (200 mg/kg i.p. or 0.1-0.2 ml at 10 mL/kg in 0.9% NaCl).

After 2-3min, verify that the animal is completely anesthetized by checking for absence of response to hind paws / tip of tail pinch using nails or small tweezers.

Once fully anesthetized, affix the animal to a silicone dissection pad (dissection pins through paws) in a splay position with ventral side up. Place silicone pad on a grid panel over a container for blood and PFA collection. Tilt the grid/pad slightly so that blood/PFA flows backward off the pad into the container. Work inside a chemical fume hood to prevent PFA inhalation.

Cut skin transversally at the base of the thorax.

Then cut muscle transversally at the same level.

Slightly lift the thorax by pulling on the xiphoid process (white tip of the thoracic body). Do not lift too much to not damage inner organs.

Cut the diaphragm with fine scissors (Be careful to not puncture the heart or lungs at this step).

Cut the thorax laterally on both sides (Be careful to not puncture the heart or lungs at this step too), pull backward, and fix thorax to pad with a needle on the left side of the animal.

Clear heart from connective tissues.

Place butterfly needle (connected to perfusion pump – prefilled with ice cold 1X PBS) in the tip of the left ventricle (darker and longer side of the heart – prominent tip). Be careful to not puncture through the right ventricle or the left atrium by going too deep. Cut the right atrium (darker atrium) to allow blood/perfusate to escape circulation.

Clamp the butterfly needle in place using a hemostat (or hold in place and clamp in next step).

Start the pump using a flow rate of 5-6mL/min. Perfuse with 10mL of ice-cold 1X PBS (0h 2m 0s).

Blood should come out of the right atrium with a regular flow (If you see clear liquid right away, you might have punctured through the heart – can try to slightly pull back the needle, but you may have a failed perfusion). Clamp the butterfly needle in place if you did not in previous step.

When blood has been depleted (~2min) switch to ice-cold freshly made 4% paraformaldehyde (PFA) 7.4 for 0h 5m 0s to 0h 8m 0s(30-50mL).

Remove brain or other tissue of interest.

Post-fix tissue submerged in 4% ice-cold PFA for 2h 0m 0s, , or as appropriate for intended use.