Perfusion Protocol

Supplies

10% formalin or 4% paraformaldehyde (depending on experiment)

Dulbecco’s phosphate buffered saline with 0.2% heparin

2 Beakers

Perfusion pump

2 strips of pump tubing (about 12-16in each)

Plastic stopcock

Needle 22G1

Autoclave tub 

Grate 

Timer

Tubes for tissue collection

Scissors

Tweezers

Bottle of 70% EtOH

 

Procedure

  1. Set up all supplies in the hood since you are working with formalin. Label your beakers “PBS w/ heparin” and “Formalin or PFA” and fill to the top with appropriate solution. Place the grate in the tub as this will be used for blood collection and a “stage” for the perfusion. The pump should be to the left of the tub and the beakers should be located behind the pump. See picture below for reference. 
  2. Plug in the perfusion pump, hook up the pump tubing to each side of the flow regulation tubing, place the stopcock and needle on the “mouse end” of the tubing, and set the perfusion pump to 50.
  3. While waiting on your mouse to be anesthetized (either by anesthetic overdose or carbon dioxide overdose), place the end of the pump tubing into the PBS beaker and fill all the way until it reaches the needle. 
  4. Once your mouse is anesthetized, place the mouse onto the grate and spray thoracic/abdominal area with 70% EtOH. Cut open thoracic cavity to expose the heart. 
  5. Use the tweezers to hold the heart steady while inserting the needle into the left ventricle. Do not insert the needle too far or you will puncture through the heart and the mouse will not perfuse well. Once the needle is placed, cut the right atrium with the scissors.
  6. Press the start button on the pump and run the PBS with heparin for 7 minutes. Continuously monitor the needle placement and the mouse during this time as the needle sometimes slips out. Also monitor the solution levels in the beakers and refill as necessary.
  7. After 7 minutes of PBS with heparin, push the stop button on the pump. Switch the tubing from the PBS with heparin to the formalin or PFA. Press the start button on the pump and set a timer for 7 minutes once you see the formalin or PFA reach the mouse (the mouse is still getting PBS for about 10 seconds and will start to “tense up” or move a little once the formalin reaches the heart). It is very important not to leave the pump running while you switch the tubing or you will introduce air into the line and pump air into the heart, which could damage it and cause a bad quality perfusion. 
  8. Monitor the needle placement especially at the beginning of the 7 minute time period because, as the mouse’s muscles tense up, the needle can slide out of the heart. Monitor the needle placement and solution levels during the entire 7 minute period. 
  9. Once the 7 minute formalin or PFA step is done, pull the mouse off the line and drain the rest of the formalin from the line into the drainage tub. Make sure to refill the tubing with PBS before moving on to the next mouse perfusion.
  10. Once all of the perfusions are complete, empty the PBS beaker into the sink and fill with warm water. Run this through the pump while you clean up. This is VERY IMPORTANT, as the tubing attachment on the pump will clog with salt from the PBS and either reduce your flow rate or clog completely. 
  11. For cleanup, empty autoclave tub (mixture of blood, PBS, and fixative) into the formalin/PFA waste collection bottle under the hood. Use a funnel for easier pouring. Any fixative left in the beaker must also go into this collection bottle. Rinse all of the tools and the grate in the sink and scrub with toothbrush. Hang grate up to dry and put tools on paper towel to dry next to sink. Rinse autoclave tub thoroughly and place into hood to dry. Make sure to also rinse CO2 tank cage and place face down in hood to dry.