Mouse anti ctbp2 antibody

All steps were performed at room temperature unless otherwise indicated. Dissected organ of Corti explants were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min, followed by permeabilization and blocking with PBT1 (0.1% Triton X-100, 1% BSA, and 5% heat-inactivated goat serum in PBS, pH 7.3) for 30 min. Samples were then incubated with rabbit anti-CDK5 antibody (Santa Cruz, Cat. No. sc-173, 1:100 diluted) or rabbit anti-MYO6 (Cell Signaling Technology, Cat. No. 9200, 1:50 diluted) in PBT1 overnight at 4°C, followed by incubation with Alexa Fluor^® 488 donkey anti-rabbit secondary antibody (Invitrogen, Cat. No. A21206, 1:200 diluted) in PBT2 (0.1% Triton X-100 and 0.1% BSA in PBS) for 1 h. After that, samples were incubated with TRITC-conjugated phalloidin (Sigma-Aldrich, Cat. No. P1951) in PBS for 30 min, then mounted in PBS/glycerol (1:1) and imaged with a confocal microscope (LSM 700, Zeiss, Germany). For CtBP2 staining, samples were incubated with mouse anti-CtBP2 antibody (BD, Cat. No. 612044, 1:100 diluted) in PBT1 overnight at 4°C, followed by incubation with Alexa Fluor^® 568 goat anti-mouse IgG1 (Invitrogen, Cat. No. A21124, 1:200 diluted) in PBT2 for 1 h and DAPI (Gene View Scientific Inc.) in PBS for 1 h.

The immunohistochemistry method to assess the number of synapses per inner hair cell (IHC) has been extensively described [14 (link)]. Briefly, the presynaptic IHC ribbons were identified using a mouse anti-CtBP2 antibody (1:500; BD Biosciences, San Diego, CA). Glutamate receptors were labelled with a mouse antibody raised against the C-terminus of the GluA2 subunit, IgG2a (1:200, Millipore, Billerica, MA). A 3D, custom algorithm was used to detect the juxtaposition of pre- and post-synaptic structures in stacked confocal images.