Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging

- First, place a euthanized mouse on a dissection board. Right above the abdomen, cut through the skin and peritoneum. Puncture the diaphragm and cut along the ribs and rib cage till the trachea. Create a small incision in the trachea. Gently insert a needle inside the trachea without injuring the carina and fasten it with forceps. Inject low melting point agarose at normal body temperature inside the lungs. Slowly remove the syringe and keep the needle inside to prevent the backflow of agarose.

Pour cold PBS on the lungs to promote agarose gelling. Remove the needle and fasten the trachea with forceps to avoid backflow of non-solidified agarose. Cut open the sternum, hold the trachea with forceps, and cut it using scissors. Pull out the lungs from the chest cavity. Separate the lobes in the media. Place inside imaging wells under physiological conditions for microscopy. Agarose preserves lung morphology at physiological temperatures that help to observe live cellular interactions. In the following protocol, we will demonstrate ex vivo live imaging of lungs to study interactions between cancer cells and stromal cells.

- To prepare the lungs for ex vivo live imaging, begin by immobilizing the mouse on a polystyrene foam lid covered with a piece of soaker and sterilize the animal with 70% ethanol. Next, using surgical scissors, make a transverse epigastric incision through the skin, followed by a similar incision through the peritoneum. Then, moving the dissection board into a vertical position, cut the descending aorta so that the blood pools into the abdomen and not into the chest cavity.

Snip a small opening in the diaphragm to release the vacuum, then cut along the rib cage to excise the diaphragm and to visualize the lungs. Using surgical scissors, cut the skin up to the trachea over the rib cage, leaving the ribs intact. Then separate the skin from the rib cage and remove the surrounding connective tissue to expose the trachea, taking care not to damage the trachea itself. Snip a small opening approximately 1 millimeter in diameter in the exposed trachea parallel to the cartilaginous rings as close to the larynx as possible, without cutting completely through the trachea.

Then gently insert a 20 gauge needle 4 to 5 millimeters into the trachea without any counterforce. The end of the needle should be visible through the trachea. Fill a syringe with 400 microliters of 37 degree Celsius 2% low melting temperature agarose, and then stabilize the needle with forceps. Keeping the dissection board vertical, slowly instill the entire quantity of warm agarose through the needle into the lungs. Once the lungs are inflated, detach the syringe while keeping the needle inside the trachea and pour approximately 50 milliliters of 20 degrees Celsius PBS over the lungs to help set the agarose.

When the agarose has solidified, remove the needle and use forceps to close the trachea to prevent the leakage of any non-solidified agarose. Now, open the chest further by sternotomy and grasp the trachea with forceps. Then, using a pair of surgical scissors, cut through the trachea completely. To remove the lungs, gently pull the trachea up, cut away the connective tissue and esophagus, and pull the lungs completely out of the chest cavity.

Immerse the tissue in 37 Degrees Celsius RPMI 1640 to wash off the excessive blood. Using scissors and forceps, cut the lobes' main stem bronchus at the hilum and gently separate the lung lobes. Transfer the lobes into one well of a 24-well imaging plate flat surface down and cover the tissue with 100 microliters of 37 degrees Celsius RPMI 1640. Place several 15 milliliter circular microscope cover slides on top of the lobes to prevent them from floating, and pour warm PBS into the surrounding wells to prevent the medium from evaporating. Then insert the 24-well plate into the climate chamber, and transfer the chamber to the confocal microscope stage.