Transcardial Perfusion in Mouse

Make mixture of ketamine:xylazine:acepromazine (4:2:1) sufficient for anesthesia of all mice (~30µL/ 20gmouse). Record ketamine used in controlled substance log book.

Apply anesthesia to one mouse via intraperitoneal injection, and place mouse in bucket long enough for anesthesia to take effect. Apply a hard toe pinch until mouse no longer reacts, ensuring that the mouse can no longer feel pain before proceeding.

Place mouse, abdomen-up, on Styrofoam block wrapped in lab mat. Spray mouse abdomen with 70% ethanol. Grasp skin below ribcage with forceps and cut skin with scissors from middle up either side towards the armpits, cutting through ribcage.

Remove pericardium and peripheral fat to expose heart. Hold back ribs with hemostats.

Place blunted butterfly needle into left ventricle and cut right atrium. Push PBS through syringe by using perfusion pump at 2 mL/minute rate.

After perfusion is complete, remove brain, spinal cord and other desired parts and transfer to fixative.

Weigh PFA under the hood and using bench pads.

Heat and stir (in chemical hood).

Lets dissolve the PFA around 60°C (use the thermometer). Do not let the temperature exceed 65°C(remove from hot plate temporarily if necessary).

Once dissolved but still foggy, 7.4 with NaOH (it should make the PFA clear).

Prepare a big filter funnel using Whatman filter paper.

Filter the 4% PFA into a liter bottle/w lid.

Store the PFA covered at4°C.