Animal Trans-cardial Perfusion Technique¶
Contributed by Luke Hammond, QBI, The University of Queensland, Australia
Animal Trans-cardial Perfusion Technique
- Animal ethics approval
- Training in the safe handling of laboratory animals
- Training in animal perfusion
Pre-perfusion buffer (for washing to clear blood vessels and open up the capillaries) Sodium Nitrite 0.01% in PBS pH 7.4 Fixative4% Paraformaldehyde +/-0.25% Acrolein (other fixatives can be used here) Anesthetic see “NHMRC Guidelines to promote the well-being of animals used for scientific purposes”
- Set up the perfusion rig with fresh filtered solutions each time and ensure that all tubing is clean and free of blockages.
- Anesthetise the animal checking the foot pinch reflex to make sure it is fully anesthetised.
- Secure the animal on its back.
- Cut open the pericardium
- Pull the heart down to reveal the ascending aorta and insert the catheter into the ascending aorta.
- Clamp the heart at right angles to the catheter.
- Cut the right atrium.
- Make sure the pericardium does not occlude the venous outflow.
The principle of perfusion is to start at a rate equivalent to cardiac output.
- Begin the perfusion by running the wash solution through to open the capillaries and wash the erythrocytes out of the tissue.
- Once cleared switch to the fixative.
- The run through time for the fixative should be 20 to 30 mins.
- Dissect out the brain and post fix for 24 hr (this may vary depending on the sensitivity of the antigen).
This method is based, with permission, on an original protocol available here.