India Ink Inflation: A Staining Method to Visualize Tumor Nodules

Begin by placing a euthanized mouse ventral side up on a surgical bed and securing the limbs of the animal. Using scissors, cut along the midline through the rib cage up to the salivary glands. Place a pipette tip underneath the trachea to elevate and isolate it. Rotate the surgical bed 180 degrees. Insert a syringe needle containing an India ink stain into the trachea and inject the ink into the lungs until you feel resistance.

Cancer cells secrete a large amount of lactic acid that generates a strong negative charge on the cell surface. When these cells are stained with India ink, an acidic stain, the cell surface repel the stain and remain unstained.

Use scissors to cut the trachea and slide a pair of tweezers under the lungs. Gently pull the lungs out of the animal's body. 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. The tumor nodules appear white against the dark background.

In the following protocol, we will perform the India ink inflation technique to validate lung metastasis.

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. Spray the mouse with 70% ethanol. 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. Identify the trachea. Then, use forceps to remove the surrounding tissues.

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. Keeping the trachea elevated, rotate the dissecting platform 180 degrees. Then, insert a half cc 27-gauge tuberculin syringe into the trachea and completely inflate the lungs with India ink until a strong resistance is felt.

Then, using a pair of scissors, cut the trachea. Holding the mouse in place with one forceps, insert another forceps under the lungs and pull the lung lobes out of the mouse. Rinse the lungs briefly in a beaker of water and transfer the tissue into a fume hood. Then, place the lungs in a glass scintillation vial containing 3 milliliters of Fekete's solution, capping the vial to prevent the solution from evaporating. After a few minutes, transfer the lungs into a Petri dish and count the number of single, white, metastatic tumor nodules.