To begin, securely hold the head of the euthanized newborn mice in place. Using micro operating scissors, open the scalp along the sagittal suture. Then, separate and immobilize the scalp bilaterally with the fingers.
Use scissors to sever the cranium and flip it forward to access the skull. Employ a small periosteal elevator to extract the brain and then use the tip of the scissors to gently scrape the brain, revealing the base of the skull. Now, examine the bilateral temporal bones at the skull's base.
Employ scissors to incise the base of the skull along the midline. Then, scrape off the skin and eliminate any unnecessary bone. Preserve and transfer the temporal bones to 35-millimeter sterile Petri dishes filled with fresh HBSS.
Next, use a pair of 5 number pointed forceps to eliminate the bola and surrounding tissue from the petrous portion of the temporal bone. Hold the forceps with one hand to stabilize the semi-circular part of the temporal bone. Use the other hand to insert the lower tip of the forceps into the round window niche, separating the lateral bone of the cochlea from the scale of vestibule.
Carefully remove the petrous portion of the temporal bone without contacting the Organ of Corti epithelium. Then, meticulously detach and extract the bony labyrinth of the cochlea, starting from the basal to the apical end. Using 5 number pointed forceps, carefully micro isolate the sensory epithelium of the Organ of Corti from the modiolus.
Next, employ micro-operating forceps to hold the spiral ligament, and gently separate it from the stria vascularis. Transfer the clean auditory epithelium, using a 200 microliter pipette, to a three millimeter sterile culture dish containing HBSS. Place the isolated auditory epithelium in a 100 millimeter sterile Petri dish filled with HBSS for the subsequent preparation steps.
