India ink staining protocol lungs9/1/2023 ![]() Cap the vial to prevent the solution from evaporating. 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).Rinse the lungs briefly in a beaker containing water. Use forceps to hold the mouse and insert another set of forceps under the lungs and pull the lung lobes out of the mouse. Use a pair of scissors to cut the trachea.Completely inflate the lungs with ink until a strong resistance is felt. 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. Rotate the platform holding the mouse 180°.Holding the tip with one hand, gently lift the trachea up and away from the body. Thread a pipette tip underneath the trachea.Use forceps to remove tissues surrounding the trachea to expose the trachea. 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.Pin the legs to ensure unobstructed access to the trachea. Place the sacrificed-mouse on its back on a polystyrene board.Wait for 3 more minutes and remove the filter top. Turn off CO2 cylinder control and the flow meter. 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. Set the flow rate at 2 L/min and keep CO2 on for at least 10 min. Turn on the compressed CO2 gas cylinder (100%) that is connected to the CO2 filter top. Remove the cage filter top and replace with the CO2 filter top.When housed with other animals, relocate those mice not being euthanized to a different cage. Keep mice in their existing home cage for euthanasia.This protocol use lung metastasis as a example. 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. NOTE: To validate the luciferase live tumor imaging results, perform India ink inflation of tumor-bearing mouse lungs to quantify tumor nodules. Validation of Lung Metastases Using India Ink Inflation of Tumor-bearing Lungs After staining, the lungs are fixed in Fekete's solution to observe tumor nodules in the lungs. This video describes india ink inflation-a staining method-to quantify tumor nodules in tumor-bearing mouse lungs. An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis.
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