Cochleae were dissected, fixated, and stained with primary antibodies against presynaptic C-terminal-binding protein 2 (anti-CtBP2, #612044, BD, Franklin Lakes, NJ) and postsynaptic glutamate ionotropic receptor AMPA type subunit 2 (anti-GluA2, #MAB397, Millipore, Burlington, MA). After washing, samples were stained with appropriate secondary antibodies and a marker against F-actin (phalloidin 405, #A30104, Thermo Fisher Scientific, Waltham, MA) to visualize HCs. After washing the sensory epithelium was dissected out from the cochlear tissue, cut into sections and flat mounted in a tonotopic manner on slides.
Ribbon synapses and OHCs were imaged with 40× and 20× oil immersion lenses, respectively, at an SP5 confocal microscope (Leica, Wetzlar, Germany). Phalloidin-stained OHCs were counted manually with ImageJ (version 1.53t, NIH) and represented per µm length of the sensory epithelium. OHCs were considered lost when both stereocilia bundles and cuticular plates were absent. For the scoring of ribbon synapses, each punctum with colocalized CtBP2 and GluA2 signal was scored as an intact, connected synapse and presented as synapses per IHC (58 (link), 59 (link)). A minimum of four z-stacks were analyzed per cochlea and synapses from at least ten IHCs per z-stack quantified.
Ribbon synapses and OHCs were imaged with 40× and 20× oil immersion lenses, respectively, at an SP5 confocal microscope (Leica, Wetzlar, Germany). Phalloidin-stained OHCs were counted manually with ImageJ (version 1.53t, NIH) and represented per µm length of the sensory epithelium. OHCs were considered lost when both stereocilia bundles and cuticular plates were absent. For the scoring of ribbon synapses, each punctum with colocalized CtBP2 and GluA2 signal was scored as an intact, connected synapse and presented as synapses per IHC (58 (link), 59 (link)). A minimum of four z-stacks were analyzed per cochlea and synapses from at least ten IHCs per z-stack quantified.
