The overall goal of this confocal live imaging method is to directly observe the cellular processes, the drive's secondary palate fusion during cranial facial development. Begin by separating the head from the body of the embryo under a dissecting microscope in a petri dish containing fresh, room temperature PBS. Grasp the head with one pair of fine number five forceps and use a second pair of forceps to remove the upper brain, cutting away any extra tissue outside of the palatal shelves with a second pair of forceps.

Remove the mandible carefully without damaging the palatal shelves. When the mandible has been detached, carefully remove the tongue and confirm the presence of a robust reporter signal in the midline epithelia seam. It's important not to damage it, palatal explained during dissection to keep the midline epithelia seam intact.

Next, remove the hind brain, brain stem and both maxilli with the adhered palatal shelves. Cut the anterior upper jaw tissue, keeping both the primary and secondary palates intact. Remove the nasal septum on the nasal side of the explant to expose the midline epithelia seam and confirm that the epithelia layer between the tissue is intact.

Now, add live imaging medium to a 35 millimeter glass bottom dish and place the dissected palatal shelves, oral side down in the dish as close to the glass bottom as possible. Then, place dry ice pellets on a conductive surface near the culture dish to expedite the solidification of the agarose within the imaging medium. When the agarose is semi-solid, mount the culture dish in an inverted confocal microscope equipped with a 37 degree celsius incubation chamber and use a syringe to apply petroleum jelly between the culture dish and the culture cover to prevent evaporation of the imaging medium.

Select a low magnification objective to locate the region of interest. Then, select a high magnification objective and scan multiple z stacks with five micron steps for 16 to 24 hours, using the appropriate laser excitation. Live imaging of palate explant culture reveals multiple cellular processes that mediate palate fusion, with the initial contacts between the two epithelia cells being made by membrane protrusions.

When the two epithelia layers meet, they form a multi cell layered midline epithelia seam, followed by intercalation to generate an integrated single cell layer, midline epithelia seam through epithelia convergence. The epithelia cells in deeper z levels are also progressively displaced to the oral side of the midline epithelia seam, contributing to the epithelia convergence process. Posterior directed cell migration is observed in middle palate midline epithelia seam.

With epithelia cell extrusion events observed during palate fusion, suggesting that the epithelia cells in the midline epithelia seam can be removed by this active process. This integrated epithelia seam will ultimately break down to achieve confluency. Further, filamentis actin becomes enriched at the border between the epithelia and mazenkemo areas, forming a cable like structure along the anterior posterior axis.

Software can be used to track specific cellular behaviors of interest, during palate fusion with the best method for cell tracking depending on the reporter signal used in the experiment.