The perfusion technique provides rapid and uniform fixation to all individual cells present in tissue. A 4 percent paraformaldehyde fixative is passed via the vascular system to obtain tissue of interest. The main target site for drug administration in a particular organ can be identified with the help of this technique. Perfusion has also helped scientists to understand the structural and functional organization of the human brain.
Instructions
1. Prepare fresh 4 percent paraformaldehyde by mixing 40 g of paraformaldehyde in 800 ml of water. Heat the solution to 60 C while stirring. Cool the solution to 4 C. Adjust the pH to 9.5 with the help of a pH meter. For adjusting pH, add glacial acetic acid to obtain the desired pH.
2. Set up the perfusion pump; attach all the glass parts and a perfusion needle. Run 100 ml of tap water through the tubing of the perfusion pump to remove any residue from the glass parts. Place a beaker of fixative in an ice box.
3. Open the end of the perfusion tube and place it in a fixative beaker. Scale the volume of solution according to the size of the animal. Now adjust the perfusion pump valve to a slow, steady drip. Close the valve. Fill the perfusion apparatus with 0.9 percent (weight/volume) saline solution.
4. Set up the surgery instruments near the operating table. Give an anesthesia injection to the animal according to its body weight (ketamine 0.5 mg/kg). Place the animal on the operating table with its back down.
5. Make a cut with a scalpel, along the length of the diaphragm. With the large scissors, blunt side down, cut the connective tissues at the bottom of the diaphragm. Expose the heart. Hold the beating heart with the forceps and insert a needle into the left ventricle.
6. Open the valve of the perfusion pump to allow the flow of 0.9 percent saline solution. Make sure the solution is flowing freely and the animal is alive. The heart of the animal should be continuously beating. Once the blood is replaced by the saline solution, change the solution to 4 percent paraformaldehyde.
7. Stop the perfusion and dissect a tissue of interest. Keep tissue in the same medium at 4 C.
Tags: perfusion pump, percent paraformaldehyde, saline solution, glass parts, operating table