In situ Hair Cell Quantification

Benefit: In situ observation of the sensory epithelium for hair cell quantification helps prevent dissection-induced tissue damage.

Procedures:
Step 1: A fixed cochlea is placed in PBS for partial dissection. The apical bony shell of the cochlea facing the auditory bulla is removed using a fine-tip diamond drill to expose the apical section. The cochlea is stained with fluorescence-labeled phalloidin in 10 mM PBS for 30 minutes at room temperature. The stained tissue is photographed in situ using an epifluorescence microscope equipped with a digital camera.

Step 2: After imaging the hair cells in the apical region, the apical turn of the sensory epithelium is collected for further procedures. The bony shell within the auditory bulla covering the middle and basal turns of the cochlea is removed to expose those regions. The tissue is stained again with fluorescence-labeled phalloidin in 10 mM PBS for 30 minutes at room temperature. The cochlea is then photographed to capture images of hair cells in the middle and basal regions. Hair cell images are collected section by section from apex to base.

Step 3: Using Adobe Photoshop, the sections are stitched together to form a panoramic view of the sensory epithelium. The number of missing outer hair cells is quantified along the cochlear spiral at 150-µm intervals, and the results are presented as a cochleogram.