Sean Lauber:Broncho-alveolar Lavage
This procedure explains how to sacrifice the mouse (by bleeding/asphyxiation). Once the mouse is bled and the diaphragm is cut back the mouse is likely dead and the anasthetic nose tube can be removed. Before beginning the procedure be sure to prepare cannulas: 21 gauge needles fitted with 0.58 mm polyethylene tubing (Itramedic PE50 NO. 427411). These cannulas will be inserted into the trachea and then secured using short pieces of thread (be sure to have thread on hand). This cannula can then be fitted with a syringe and PBS can be used to fill the lung and then retracted for collection. This is the broncho-alveolar lavage.
This same procedure is required to isolate the lungs for formalin fixation.
- Anesthetize mouse using isoflurane
- Using a nose cone stuffed with isoflurane-soaked cotton, remove the mouse to the work bench and maintain anesthesia
- Soak the mouse in EtOH and make an incision in the abdomen
- Tear back the skin to expose the peritoneum
- Cut back the peritoneum to expose the guts
- To bleed the mouse, locate the renal artery and cut it (collect blood if needed)
- Cut back the diaphragm, being sure not to cut the lungs
- Cut out the rib cage, making sure not to cut the lungs
- Pull the rib cage free while exposing the trachea
- Remove excess tissue around the trachea
- Tie a piece of thread loosely around the trachea
- Make an incision in the trachea small enough to permit a cannula. Be sure not to cut the trachea all the way through. If you do this there is little hope for continuing. Just "nick" the trachea.
- Slide the cannula in (this might take some getting used to - I like to hold the animal upright and insert it from above)
- Secure the cannula using the piece of thread (make sure it is very tight - use the forceps to get a good grip)
- Cut the trachea free from above the point of cannula insertion
- Remove excess tissue from below the trachea while pulling the lungs free
- Instill 500 μL of sterile PBS (RT) into the lungs slowly
- Bounce the lungs around for several seconds before removing the liquid. Massage the lungs to help drive the liquid out. Place the liquid on ice.
- Repeat one more time to collect about 500-700 μL of BAL.