Mouse anti ctbp2

Primary antibody were used as follows: rabbit anti-Myosin7a (1:200; Santa Cruz Biotechnology), mouse anti-Ctbp2 (1:200; BD Transduction Laboratories), and mouse anti-Gria2 (aka GluR2/GluA2; 1:2000; Millipore). Secondary antibodies were all purchased from Jackson Laboratories and were used as follows: goat anti-mouse 488 (1:500), goat anti-mouse 594 (1:500), donkey anti-rabbit 647 (1:200).

The following primary antibodies were used: mouse anti-Tubulin beta III isoform C-terminus, clone TUJ1 (1:500; Millipore; RRID:AB_2210524), goat anti-Oncomodulin (Ocm) antibody (1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Foxo3 (1:1000; Thermo Scientific; RRID:AB_2544621), rabbit anti-Myosin7a (Myo7a) (1:200; Proteus; RRID:AB_10013626) mouse anti-Ctbp2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-Gria2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875). The following secondary antibodies, all purchased from Jackson ImmunoResearch, were used: Donkey Anti-Mouse 488 (1:500; RRID:AB_2340849), Donkey Anti-Rabbit 594 (1:500; RRID:AB_2340622), Donkey Anti-Rabbit 647 (1:200; RRID:AB_2340625), Donkey Anti-Goat 647 (1:200; RRID:AB_2340438), Alexa 594 Goat Anti-Mouse (IgG1, 1:500; RRID:AB_2338885), Alexa 488 Goat Anti-Mouse (IgG2a, 1:500; RRID:AB_2338855).

Animals were decapitated after anaesthesia, and the temporal bones were removed and fixed for 2 h in 10% formaldehyde in phosphate-buffered saline (PBS). After the samples were rinsed in PBS, the basilar membranes of the cochlea were dissected out for immunostaining. Permeabilized with 0.3% TritonX-100 (Sigma-Aldrich) for 30 min and blocked with 10% normal goat serum (Jackson) for 1 h; the tissues were incubated overnight at 4°C with the following primary antibodies: rabbit anti-myosin 7a (1 : 300, Proteus Biosciences); phalloidin 594 (1 : 500, Thermo Fisher); mouse anti-CtBP2 (1 : 300, BD); and chicken anti-NF200 (1 : 200,CHEMICOM). After rinsing with PBS, the samples were incubated in fluorescently labelled secondary antibodies (Alexa Fluor 488 and 568, Invitrogen/Molecular/Thermo Fisher) for 1 h at room temperature. Nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI) (AppliChem).

Rabbit anti-calbindin (horizontal cells; Swant; CB38a; 1:1,000), Rabbit anti-cone arrestin (cones; Millipore; AB15282; 1:5,000), rabbit anti-PSD95 (synapses; Cell Signaling Technology; 1:1000), mouse anti-CTBP2 (synapses: BD Biosciences 1:1,000), peanut lectin (cone terminals: Millipore 1:2,000). Goat anti Bassoon (synaptic stains; Santa Cruz Biotechnology, Santa Cruz, CA; sc-18565; 1:400), and DAPI reagent (mixed into the second wash after incubation with secondary antibodies at a dilution of 1:50,000 of a 1 mg/ml stock). Secondary antibodies were acquired from Jackson Immuno Research and used at a concentration of 1:1000. Tissue was stained as previously described [40 (link)]. Briefly, antibodies were diluted in a blocking solution of 0.1% triton (sections) or 0.4% triton (whole retinas) supplemented with 7% normal donkey serum in PBS. Sections were blocked for 20 minutes in blocking solution and then incubated in primary antibody overnight at 4° C or at room temperature for one hour. Whole retinas were blocked for two hours in blocking solution incubated at 4° C for four days. Sections were washed 3 x for five minutes in PBS, while the whole retina washes were carried out for one hour. Tissue was incubated in secondary antibody diluted in blocking solution, as performed for primary antibodies, washed and mounted in 80% glycerol.

For whole-mount beta-III Tubulin (Tuj-1), Myosin6 (Myo6), C-terminal binding protein 2 (CtBP2), Cleaved Caspase-3 (CC3), and Parvalbumin (PV) staining, mouse heads at desired time points were fixed with 4% PFA for 2–24 hours. Fixed cochleae were dissected out and permeabilized with 0.5% TrionX-100 followed by incubation in 10% serum blocking buffer for at least 1 hour at room temperature. Incubation of primary antibody was carried out for overnight at 4 °C, followed by Alexa-conjugated secondary antibodies (1:1000, Invitrogen). For cross-section staining, mouse heads were fixed with 4% PFA for overnight. Fixed inner ear bone structure was dissected out and cryoprotected with sucrose. Tissue was then embedded in OCT (Tissue-Tek) and cut into 35-μm sections using a vibrotome (Leica). Sections were later stained using Tuj-1, Myo6, and PV as the whole-mount staining procedure. Antibodies used in this study and their dilution were as followed: Alexa488-conjugated mouse anti-Tuj1 (1:300; Covance), rabbit anti-PV (1:300; Swant Inc.), rabbit anti-Myo6 (1:500, Millipore), mouse anti-CtBP2 (1:250, BD Biosciences), rabbit anti-CC3 (1:300, Cell Signaling Technology).