Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

After euthanizing an adult mouse, use large sterile dissection scissors to cut the cheeks and break the jaw. Then, lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity. Cut the tongue out and collect it in sterile ice-cold dPBS with calcium and magnesium.

Under a dissecting microscope, remove and discard the anterior tongue by cutting just anterior of the intermolar eminence with a razor blade. Then, using a delicate task wipe to remove any hair and excess liquid from the posterior tongue.

Next, fill a 1-milliliter syringe with 200 to 300 microliters of injection enzyme solution and insert a 30-gauge, 1/2 inch needle just above the intermolar eminence until just interior of the circumvallate papillae or CVP. Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues. Withdraw the syringe slowly and continuously from the tongue as the solution is being injected.

Incubate the tongue in sterile calcium/magnesium-free dPBS at room temperature for precisely 33 minutes. Using extra fine dissection scissors, make small cuts in the epithelium bilaterally and just anterior of the CVP. Then, gently peel the epithelium by lifting it with fine forceps. Once the trench epithelium is free of the underlying connective tissue, place it in a 2-milliliter microcentrifuge tube pre-coated with FBS.