The overall goal of this protocol is to study the uptake of regular dextran fluorescently labeled with Texas Red in live hair cells with functional mechanotransduction channels. The main advantage of this technique is that the three kilodalton Texas Red dextran can be used to assess mechanotransduction channel function in live hair cells. To harvest the cochlea, place the skull of a postnatal day six mouse into a 60 millimeter culture dish containing Leibovitz's L-15 medium and use surgical scissors to make a superficial cut from the anterior end to the posterior end of the skull and across the external auditory canals.
Fold the skin toward the nose to expose the cranium and make an incision from the back to the front of the skull and across the eye line. Separate the skull into two halves and use a small spatula to remove the brain. When the temporal bones can be observed, use small scissors to cut around these bones to excise the tissue.
Submerge the temporal bones in the 35 millimeter dish containing fresh L-15 medium and place the dish under a stereo microscope equipped with a wide field eyepiece and an external alternating current halogen light source. Use forceps to remove the surrounding cochlear tissue, semi-circular canals and vestibular organs. Once the cochlea has been dissected and placed in a new dish with fresh L-15 media, use surgical forceps to make one puncture wound on the round window and one puncture at the apical cochlear region.
When all of the cochleae have been harvested, place the tissue into a well of a nine-well glass depression plate containing 200 microliters of fresh L-15 medium. For dextran labeling of the isolated tissues, replace the medium from each sample well with 200 microliters of fresh L-15 medium supplemented with two milligrams per milliliter of the fluorescence conjugated dextran of interest. Incubate the samples for two hours at room temperature and 25 rotations per minute in a three-dimensional shaker at a tilted angle of 25 degrees protected from light.
At the end of the incubation, wash the tissues one time with medium and one time with HBSS for two minutes per wash. After the second wash, fix the specimens in 4%paraformaldehyde for 30 minutes at room temperature followed by two quick and gentle washes in HBSS. After the second wash, use fine tip forceps to remove the cochlear bone, the spiral ligament, and the tectorial membrane and wash the isolated organ of Corti in HBSS.
Take extra care when removing the transparent tectorial membrane since the tissue on the hair cells can be easily damaged during this step. To label the F-actin, permeabilize the samples in 0.5%Triton X-100 in PBS supplemented with 100 to 200 dilution of fluorescently labeled phalloidin for 30 minutes at room temperature protected from light. At the end of the incubation, wash the tissues two to three times in fresh HBSS per wash as demonstrated followed by a single wash in PBS.
Use an appropriate mounting medium to mount the organ of Corti tissues onto glass microscope slides hair stereocilia sides up and image the samples on a fluorescent confocal microscope using the 63X oil immersion objective. After a two-hour incubation with three kilodalton dextran fluorescently conjugated with Texas Red, organ of Corti explant from wild type postnatal day six mice demonstrate a robust and specific labeling of both inner and outer hair cells. Imaging of the hair cell stereocilia and cell bodies reveals that only those cells that have incorporated three kilodalton dextran Texas Red in their cell body also demonstrated fluorescent labeling of their stereocilia.
Importantly, a uniform fluorescent signal is observed along the stereocilia with enrichment at the tips of the shorter stereocilia rows where the mechanotransduction channel is located. Vesicle-like structures in the cell body of the hair cells and the neighboring supporting cells are also observed suggesting that dextran Texas Red can also be taken up by endocytosis. Larger 10 kilodalton dextrans also produced a vesicle-like pattern in the cell body of hair cells and supporting cells.
Notably, the presence of a calcium chelator or mechanotransduction channel blockers prevents stereocilia labeling and the uptake of three kilodalton dextran Texas Red in hair cells. The vesicle-like pattern is still observed in the presence of mechanotransduction channel blockade, however, indicating that this pattern of uptake is independent of functional MET channels. It is very important to dissect the organ of Corti tissue carefully and to confirm the proper orientation of the hair cells before mounting the tissue for imaging.
After dextran labeling of live and functional hair cells, immunostaining of the fixed tissue can be performed to identify specific cell types of proteins of interest.
