Monoclonal mouse anti ctbp2 antibody

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).

For the biolistic transfection experiments, retinas were fixed in 4% PFA in ACSF for 20–30 min. Following an overnight block containing 5% normal donkey serum (Jackson ImmunoResearch), 2% BSA (Sigma), and 0.5% Triton X-100 (Millipore) in PBS, the retinas were incubated with the following primary antibodies for 4 nights: mouse monoclonal anti-CtBP2 antibody (1:1000; BD Biosciences), anti-synaptotagmin 2 (mouse 1:300, SyT2, ZIRC), anti-PKCa antibody (mouse 1:500; Novus Biologicals) or anti-GluR2/3 (rabbit 1:500, Millipore). After washing and incubating with the appropriate secondary antibodies (Alexa, Invitrogen, 1:1000; or Dylight, Jackson ImmunoResearch Laboratories, 1:1000, conjugated fluorophores) overnight at 4°C, retinas were washed again and mounted onto glass slides using Vectashield (Vector Laboratories). The same tissue was also used for bipolar axon terminal volume analysis (see supplemental methods).