Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Three days after plating, use gravity sedimentation to collect otospheres as previously shown. Proceed to aspirate the supernatant, and gently resuspend the cells in 2 milliliters of sensory epithelial differentiation medium. Then, use a 10-milliliter large-bore pipette to transfer the otospheres to a new 60-millimeter dish. Differentiate the otospheres for 10 days, adding 2 milliliters of fresh differentiation medium to the culture every other day.

Afterwards, harvest the otospheres again through gravity, sedimentation, and aspirate the spent medium. Next, fix the otospheres in 4% paraformaldehyde in 1X PBS for 15 minutes at room temperature. Then, remove the formaldehyde solution, and wash the cells in 1X PBS containing 0.1% Triton X-100. Proceed by blocking the otospheres in 1X PBS, supplemented with 10% goat serum and 0.1% Triton X-100.

After one hour, replace the blocking buffer with that containing the appropriate dilution of Cdkn1b or Cdh1 antibody. Then, incubate the cells in primary antibody overnight at 4 degrees Celsius, and the next day, continue immunostaining using fluorescently labeled secondary antibodies, phalloidin, and Hoechst as described elsewhere.

To prepare for imaging, place mounting medium on a glass slide and then introduce the stained otospheres into the medium. Next, lay a coverslip on top of the sample. After letting the mounting medium dry overnight at 4 degrees Celsius, place the slide on the stage of an inverted microscope equipped with a 16-bit CCD camera. Using the 20X 0.75 air objective, acquire epifluorescence images by collecting data from the blue, green, red, and infrared channels.