Perfusion-fixation procedure for adult rhesus monkeys

Add glutaraldehyde to the fixative solution.

Start oxygenation of the Ringer’s solution.

Anesthetize the animal with Ketamine (10mg/kg, i.m.) or Telazol (3mg/kg-5mg/kg, i.m.) in its home cage and bring to the necropsy room.

Place an i.v. catheter.

Perform a tracheal intubation.

Inject 1mL of heparin (i.v.).

Inject an overdose of pentobarbital (25mg/kg i.v.).

Ensure the absence of all reflexes, including toe pinch reflexes and brainstem reflexes, such as corneal reflexes.

Cut the thoracic cage to provide access to the heart, clamp the descending aorta, open the pericardium and expose the heart muscle, make an incision of the right atrium and insert the perfusion needle in the left ventricle.

Artificially ventilate the animal through the tracheal tube during the Ringer’s solution perfusion.

Infuse transcardially ~300-400mL of cold oxygenated Ringer’s solution at a rate of 80-90 ml/min through a needle (14G; 1.5 inch long) inserted in the left ventricle. Let the infused solution exit the vascular system through a hole in the right atrium.

Perfuse 2.5L of fixative, starting at a rate of 80-90 ml/min for the first liter and then reduce the rate to 50 ml/min for the remaining solution.

After perfusion, fix the animal’s head in a stereotaxic frame and open the skull using a bone saw and rongeur.

Cut the brain in 10 mm -thick blocks in the coronal stereotaxic plane, remove the resulting blocks of tissue from the skull and post-fix them for 24h 0m 0s in 4% paraformaldehyde solution in PB (0.1Molarity (M), 7.4) at 40°C.

Transfer the tissue into a phosphate-buffered saline (PBS, 0.01Molarity (M), 7.4) solution.

Cut the brain in 50 µm -thick sections using a vibrating microtome or a freezing microtome and store in a -20°C freezer in an anti-freeze solution ( Please refer material section ) until further processing.