Mouse perfusion
Bryan Killinger
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Abstract
General protocol for mouse perfusion fixation.
Steps
Anesthetize mouse using mixture of ketamine and xylazine. (Ketamine = 100mg ketamine / kg bodyweight, 10mg xylazine / kg bodyweight)
Ensure deep anesthesia with paw pinch. Mouse should not respond.
Using four 26g needles, secure mouse paws to corkboard.
Make an incision along the abdomen, extending up to the rib cage.
Expose chest cavity and secure with hemostats clamped to the xiphoid process.
Insert 22g needle into the left ventricle and secure with hemostats.
Make small incision in the right ventricle.
immediately start peristaltic pump, flowing at a rate of ~8-10mL per min.
Once the perfusate runs clear, stop pump, switch to 4% PFA solution, and resume flow.
Perfuse mouse with approximately 50-100mL of 4% PFA.
Remove head with scissors, and carefully dissect out the brain.
Scull should be snipped at the base, and peeled away carefully. Care should be taken not to damage underlying brain tissues or olfactory bulb.
Place dissected brain in 20mL 4% PFA solution, overnight, at 4C.
Place in 15% sucrose PBS solution.
Once brain sinks, Place in 30% sucrose PBS solution at 4C until sectioning.
0.05% sodium azide can be included in sucrose solutions to prevent bacterial growth.
