Keeping the trachea elevated, rotate the dissecting platform 180 degrees. Next, holding a pipette tip in one hand, thread the tip under the trachea and gently lift the tissue up and away from the body. Then, use forceps to remove the surrounding tissues. Then, use a pair of scissors to make an incision along the midline of the mid abdomen of the mouse through the rib cage and up toward the salivary glands. To validate the metastasis of the primary tumor to the lung, first place the mouse in the supine position on a polystyrene board and secure the legs to ensure an unobstructed access to the trachea. In the following protocol, we will perform the India ink inflation technique to validate lung metastasis. The tumor nodules appear white against the dark background. Rinse the lungs in water and transfer them into a vial containing Fekete's solution to remove excess stain and fix the tissue in a fume hood.

Gently pull the lungs out of the animal's body. Use scissors to cut the trachea and slide a pair of tweezers under the lungs. When these cells are stained with India ink, an acidic stain, the cell surface repel the stain and remain unstained. Insert a syringe needle containing an India ink stain into the trachea and inject the ink into the lungs until you feel resistance.Д¬ancer cells secrete a large amount of lactic acid that generates a strong negative charge on the cell surface. Place a pipette tip underneath the trachea to elevate and isolate it. Using scissors, cut along the midline through the rib cage up to the salivary glands. Begin by placing a euthanized mouse ventral side up on a surgical bed and securing the limbs of the animal.