Evaluating Cochlear Hair Cell Toxicity

To evaluate the toxicity of H₂O₂, cultured organ of Corti explants were immunolabeled with a rabbit polyclonal antibody against myosin 7A (1/300, Proteus Biosciences Inc. #25-6790) and a mouse monoclonal antibody against neurofilament (NF 200, 1/600, Sigma-Aldrich #N0142) to label hair cells and spiral ganglion neurons, respectively. Hair cell counting (six cochleae per condition and per time point) was performed using standard techniques [20 (link)]. Due to the resistance of apical turn hair cells to H₂O₂ cytotoxicity, counting concerned only a 1.5-mm length of the cochlear duct at the basal turn (4 to 5.5 mm from the apex, see [12 (link)]).
The TUNEL kit (DeadEnd™ fluorometric TUNEL System, Promega #G3250) was used to identify apoptotic DNA fragmentation. The cochlear samples were counterstained with a rabbit polyclonal antibody against myosin 7A and a mouse monoclonal antibody against neurofilament. All secondary antibodies were used at a dilution of 1/1000. This included donkey anti-mouse and anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (Molecular Probes #A-21202, #A-21206, #A-10037, #A-10042). Fluorescent tags were visualized using a confocal microscope (LSM 5 Live Duo, Zeiss). In control specimens without primary antibodies, neither Alexa 488 nor 568 fluorescent tags were observed. All experiments were performed in triplicate.