Mouse anti glur2

For Western blot analyses, the following primary antibodies were used: mouse anti-PSD-95 (1:250; BD transduction), rabbit anti-CDK5 (1:500; Santa Cruz biotechnology, Dallas TX), mouse anti-Akt (1:1000, Cell signal technology, Danvers, MA), rabbit anti-p44/42 MAPK (Erk1/2) (1:1000; Cell signal technology), mouse anti-GFAP (1:500, Sigma Aldrich, Oakville, Ontario), mouse anti-syntaxin (1:10 000, Sigma Aldrich), rabbit anti-GluR1 (1:2000, Millipore, Billrica, MA), mouse anti-GluR2 (1:2000, Millipore), mouse anti-NR1 (1:2000), rabbit anti-D2R (1:500, Millipore) and mouse anti-actin (1:10 000; Millipore). Secondary antibodies IRDye 680 Goat Anti-Rabbit IgG (1:10 000; Mandel Scientific, Guelf, Ontario) or IRDye 800 Goat Anti-Mouse (1:10 000; Mandel Scientific) were then used.
For immunochemistry analysis, mouse monoclonal anti-neuronal nuclei (NeuN) (1:250; Millipore) and mouse monoclonal anti-actin (1:5000; Millipore) were used as primary antibodies. Revelation of labeling using the Odyssey imager was performed using IRDye 800 Goat Anti-mouse IgG (1:1000) and IRDye 680 Goat Anti-mouse IgG (1:1000) secondary antibodies.

Cochleae were dissected and perfused through the round window and oval window with 4% paraformaldehyde in PBS, then post-fixed in the same solution. Cochleae were dissected into half-turns for whole-mount processing. Immunostaining began with a blocking buffer (PBS with 5% normal goat or donkey serum and 0.2–1% Triton X-100) for 1 to 3 h at room temperature and followed by incubation with a combination of the following primary antibodies: (1) rabbit anti-CtBP2 (BD Biosciences, Catalog No. 612044) at 1:100, (2) rabbit anti-myosin VIIa (Proteus Biosciences, Catalog No. 25–6790) at 1:200, (3) mouse anti-GluR2 (Millipore, Catalog No. AB1768-I) at 1:2000. Lengths of cochlear whole mounts were measured and converted to cochlear frequency. Confocal z-stacks from each ear were obtained in the inner hair cell area using a high-resolution glycerin-immersion objective (63×) and ×3.18 digital zoom with a 0.25 μm z-spacing on a Leica SP5 confocal microscope. For each stack, the z-planes imaged included all synaptic elements in the x–y field of view. Image stacks were imported to image-processing software (Amira, Visage Imaging), where synaptic ribbons, glutamate receptor patches, and inner hair cells were counted.