Ex Vivo Culture and Imaging of Oculomotor Slices from Transgenic Mouse Embryos

Take a transgenic pregnant mouse, surgically isolate the uterus containing embryos, and wash it to remove residual blood.

Remove the embryos containing fluorescently labeled motor neurons and transfer them to the lid of a multi-well plate.

Add molten agarose and cool the lid to induce gel formation, embedding the embryos.

Under a fluorescence microscope, align the oculomotor nuclei and the eyes in a line and trim the agarose parallel to the line.

Fix an embryo on a specimen stage and place it in a vibratome chamber filled with a cold slicing buffer.

Using the vibratome, generate slices containing the oculomotor nuclei and eyes. The nuclei form the oculomotor nerves that control eye movement.

Transfer the slices onto culture inserts inside a multi-well plate containing a culture medium and incubate.

Using a fluorescence microscope, image at regular intervals to observe the fluorescently labeled oculomotor neurons extending axons to reach the eyes.

Before beginning the procedure, first, confirm the pregnancy by ultrasound on embryonic day 10.5 from a timed mating. Harvest the embryos, spray the abdomen of the pregnant mouse with 70% ethanol, and use scissors to open the abdominal cav