Immunostaining of Cochlear Synapses

Immunostaining was performed as previously described [16 (link)] with minor modifications. Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, Haverhill, MA, USA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4 °C overnight. After adequate decalcification by incubation in 500 mM EDTA (3–6 days), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories [87 ]. The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technology, Danvers, MA, USA). Pre-synaptic ribbons and post-synaptic densities were labelled using a monoclonal mouse anti-CtBP2 antibody (1:200, BD Biosciences, San Jose, CA, USA) and a monoclonal mouse anti-GluR2 antibody (1:2000, Sigma-Aldrich, St. Louis, MO, USA), respectively, followed by incubation with the corresponding secondary antibodies, goat anti-mouse IgG2 Alexa Fluor^® 488 and goat anti-mouse IgG1 Alexa Fluor^® 568 (1:1000, ThermoFisher Scientific, Waltham, MA, USA). Nuclei were counterstained with DAPI. The labelled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific, Waltham, MA, USA).