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 immobi