Mouse igg2 anti glua2

Mice were perfused intracardially with 4% paraformaldehyde (PFA) for 5 minutes, after which the cochleas were exposed and perfused with 4% PFA after opening the oval and round windows. The perfused cochleas were extracted, post-fixed for 2 hrs in 4% PFA, and decalcified in EDTA for 48 hrs at room temperature. They were then dissected into half-turns, and incubated in primary antibodies: 1) mouse (IgG1) anti-CtBP2 from BD Transduction Labs at 1:200 and 2) mouse (IgG2) anti-GluA2 from Millipore at 1:2000. Primary incubations were followed by 1-hr incubations in species-appropriate secondary antibodies.

Histology and imaging were performed using procedures detailed previously (Valero et al 2017 (link)). Briefly, following completion of the behavioral assays, animals were euthanized by an overdose of sodium pentobarbital (130 mg/kg), followed immediately by transcardial perfusion (2 liters 0.9% phosphate-buffered saline, PBS; 2 liters 4% phosphate-buffered paraformaldehyde, PFA). The round and oval windows were opened, cochleas perfused through the scala tympani with PFA, submerged in PFA for 2 hours, then transferred to 0.12 M EDTA for decalcification.
Decalcified cochleas were dissected into quarter turns to obtain epithelial whole mounts of the organ of Corti containing the hair cells and most of the osseous spiral lamina at each location from base to apex. Immunohistochemistry was used to label pre-synaptic ribbons (mouse IgG1 anti-CtBP2 (C-terminal binding protein 2); BD Transduction Labs; 1:200); ii) glutamate receptor patches (mouse IgG2 anti-GluA2; Millipore; 1:200), iii) hair cell cytoplasm (rabbit anti-myo7a (myosin VIIa); Proteus Biosciences; 1:200), and iv) cochlear afferent and efferent fibers (chicken anti-NFH (neurofilament-H); Chemicon; 1:1000). Tissue was incubated in species-appropriate fluorescent secondary antibody conjugates (AlexaFluor) for secondary detection.

Mice were euthanized by transcardial perfusion while deeply anesthetized with ketamine. Following a 5-min perfusion with 4% paraformaldehyde, cochleas were exposed and perfused through the oval and round windows. Cochleas were then post-fixed for 2 hrs, decalcified in EDTA for 48 hrs at room temperature, and then dissected into half-turns and incubated in primary antibodies: 1) mouse (IgG1) anti-CtBP2 from BD Transduction Labs at 1:200; 2) mouse (IgG2) anti-GluA2 from Millipore at 1:2000, 3) rabbit αMyo7a from Proteus Biosciences at 1:200 for 18 hrs. Primary incubations were followed by two 1-hr incubations in species-appropriate secondary antibodies.