Immunostaining of Mouse Cochlear Tissues

Immunostaining was performed as previously described with minor modifications.^{7 (link),9} Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, MA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4°C overnight. After adequate decalcification by incubation in 500mM EDTA (3–6 days), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories.¹² The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, MO) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technologies, MA). Pre-synaptic ribbons and post-synaptic densities were labelled using a monoclonal mouse anti-CtBP2 antibody (1:200, BD Biosciences, CA) and a monoclonal mouse anti-GluR2 antibody (1:2000, Sigma-Aldrich, MO), respectively. Following this, the tissue was incubated with the corresponding secondary antibodies, goat anti-mouse IgG2 Alexa Fluor^® 488 and goat anti-mouse IgG1 Alexa Fluor^® 568 (1:1000, ThermoFisher Scientific, MA). Nuclei were counterstained with DAPI. The labelled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific, MA).