Perfusion of Mice and Dissection of Cochlea

Perfusion of Mice (Yoash Raphael) Hazards: When working with fixative always wear gloves. Use glutaraldehyde in the hood. Dispose of all excess fixative in correctly labeled bottles in the hood and solid waste in bucket.

Pump: we infuse the buffer and then the fixative solutions with an electro-mechanical pump, in which the rate of infusion can be adjusted. For guinea pigs, we just use gravity. Mice are very small and delicate and we found that use of a pump provides better controlled pressure of infusion.

Materials: 8% Paraformaldehyde (ICC or IHC) or Glutaldehyde/ Sodium Cacodylate –SEM or TEM preparation PO4 buffer PBS Syringe pump 60cc syringes for pump (labeled buffer and fix) and tubing 1cc syringe & needle Ketamine (~ 0.05cc for mouse) scintillation vials and caps forceps small scissors lg. Scissors 2 hemostats 15cm petri dish 35mm petri dish

Preparation 1. Make fixative solution. (2% glut or -2-4% PFA) 2. Fill fix syringe with appropriate amount of fix solution. 3. Fill buffer syringe with PBS 4. Prime tubing in fix syringe then the PBS. Place PBS syringe in pump (pump should be set at 3mls/min).You always want to start the perfusion with buffer (PBS) syringe. 5. Inject mouse with Ketamine (I.P.) 6. Place a small amount of fix solution in scintillation vial (one for each mouse you do). Label cap with date and code.

Method Once mouse is down, tape all 4 limbs down on an inverted 15cm petri dish. Dissect chest area to expose heart. Clamp rib cage back with one hemostat. Clamp lower artery and vein with the other hemostat.

Snip the right atria just a little. Hold on to the heart with forceps and place perfusion needle in the lower (distal) left ventricle. Make sure the needle is not up against the wall. You must hold on to the needle during the entire perfusion. Pump buffer for 1-2 minutes or until clear fluid runs out and the heart is white. Stop pump. Hold on to needle and switch to fix syringe. Pump fixative until nose is stiff (~5-10mls). Stop pump.

Remove mouse from petri dish. Cut head off behind the ears. Cut from side of mouth to remove lower jaw. Cut upper portion of head thru the center to make two symmetrical sections. Work on one section at a time. Remove brain and cut out ear. Place into 35mm petri dish with fixative. Open apex and remove stapes. Manually perfuse fixative thru cochlea with a 1cc syringe. Place cochlea in scintillation vial and fix for 1-2 hrs on a shaker at room temperature