India Ink Inflation: A Staining Method to Visualize Tumor Nodules

1. Validation of Lung Metastases Using India Ink Inflation of Tumor-bearing Lungs

NOTE: To validate the luciferase live tumor imaging results, perform India ink inflation of tumor-bearing mouse lungs to quantify tumor nodules. 4T1 cells also metastasize to other organs and tissues (Figure 1) and therefore, histological examination of these organs to validate tumor metastasis should be performed if needed. This protocol use lung metastasis as a example.

1. Keep mice in their existing home cage for euthanasia. When housed with other animals, relocate those mice not being euthanized to a different cage.
2. Remove the cage filter top and replace with the CO2 filter top. Turn on the compressed CO2 gas cylinder (100%) that is connected to the CO2 filter top. Set the flow rate at 2 L/min and keep CO2 on for at least 10 min. If the mice are still breathing at the end of 10 min, leave the CO2 on until at least 1 min after mice stop breathing. Turn off CO2 cylinder control and the flow meter. Wait for 3 more minutes and remove the filter top.
3. Place the sacrificed-mouse on its back on a polystyrene board. Pin the legs to ensure unobstructed access to the trachea. Spray the mice with 70% ethanol.
4. Using a pair of scissors, cut along the midline of the mid-abdomen of the mouse through the ribcage and up toward the salivary glands. Observe the trachea. Use forceps to remove tissues surrounding the trachea to expose the trachea.
5. Thread a pipette tip underneath the trachea. Holding the tip with one hand, gently lift the trachea up and away from the body.
6. Rotate the platform holding the mouse 180°. Use a ½ CC 27 G 1/2 tuberculin syringe to inject India Ink (10% India ink and 0.1% Ammonium Hydroxide) into the lungs via the trachea. Completely inflate the lungs with ink until a strong resistance is felt.
7. Use a pair of scissors to cut the trachea. Use forceps to hold the mouse and insert another set of forceps under the lungs and pull the lung lobes out of the mouse. Rinse the lungs briefly in a beaker containing water.
8. In a chemical fume hood, transfer the lungs to a glass scintillation vial containing 3 ml of Fekete's solution (50% Ethanol, 6% Formaldehyde, and 3% glacial acetic acid). Cap the vial to prevent the solution from evaporating.
   NOTE: The tissues can be stored in Fekete's solution indefinitely.
9. After a few minutes, observe tumor nodules as white dots on the black lungs (Figure 1). The white tumor nodule is visible by eyes. Transfer the lungs to a dish in a fume hood and count the number of white spots. Each white spot represents a single metastatic tumor nodule.