Evaluation of Ribbon Synapses at Specific Frequency Regions of Mouse Cochlea

Begin with tubes containing pre-treated mouse cochlear turns with a permeabilizing agent and a blocking agent that prevent non-specific interactions.

Each turn detects different sound frequencies and contains hair cells that convert sound into electrical signals. These signals are transmitted via neurotransmitters in ribbon synapses to the auditory nerve.

Remove the solution and add primary antibodies that interact with proteins in hair and nerve cells at the ribbon synapses, then wash.

Incubate with secondary antibodies tagged with red and green fluorophores, which bind to their primary antibodies.

Wash to remove unbound secondary antibodies.

Place the specimen on a glass slide with a mounting medium containing nuclear dye to stain nuclei blue.

Mount the coverslip. Dry it, then use a confocal microscope to image each segment at various depths.

Ribbon synapses appear as closely positioned red and green puncta.

Count these functional puncta in each turn to correlate their response to sound frequency.

After dissection, place each cochlear turn into individual 2.5-milliliter centrifuge tubes. Block any nonspecific binding with 10% goat serum in PBS in 0.1% Triton X-100 for 1 hour at r