Mab397

Protein samples were prepared with reducing Laemmli buffer and boiled for 10 min at 95°C, and then separated on NuPAGE 4–12% Bis-Tris Mini protein gel (Thermo Fisher Scientific NP0323BOX) for 3 h at constant voltage of 90 V. After gel electrophoresis, proteins were transferred onto Immobilon-P PVDF membranes (Millipore IPVH00010) for 90 min at constant current of 0.4A. Membranes were blocked with 1x Tris Buffered Saline (TBS) with 1% Casein (BioRad, #1610782) for 1 h at room temperature in gentle shaking, and then incubated at 4°C overnight with the primary antibody in blocking buffer, rabbit anti-Tyro3 (Cell signaling 5585S) at a dilution of 1:2000, mouse anti-PSD95 (Thermo Fisher Scientific MA1-046) at a dilution of 1:2000, mouse anti-GluA2 (Millipore MAB397) at a dilution of 1:5000, rabbit anti-synaptophysin (Abcam ab16659), or mouse anti-GAPDH (Santa Cruz SC-32233) at a dilution of 1:10,000. After washing with TBS buffer containing 0.1% Tween-20, membranes were incubated with secondary antibody for 3 h at room temperature (Peroxidase AffiniPure Goat Anti-Rabbit IgG, Jackson Immunoresearch 111–036-047 or 115–006-072). After washing with TBS buffer containing 0.1% Tween-20, membranes were incubated in chemiluminescent substrate (SuperSignal, ThermoFisher 34,580) and the chemiluminescent signal was detected and recorded by exposure of the membrane to X-ray film.

P8 or P15 mice were anesthetized with an intraperitoneal injection of a lethal dose of sodium pentobarbital and perfused with 4% paraformaldehyde in 0.1 M PB, pH 7.4. Brains were removed and post-fixed in the same fixative for 24 hours at 4 °C. Cryostat coronal slices of 16 μm thickness were immersed in citrate buffer 0.01 M for heat induced epitope retrieval. Standard procedures of immunohistochemistry were performed. Primary antibodies used were rabbit polyclonal anti-GluR1 (AB1504, Millipore) and mouse monoclonal anti-extracellular GluR2 (MAB397, Millipore) antibodies. An undiluted CSF from a patient with limbic encephalitis containing LGI1 antibodies (as assessed by clinical diagnosis) was used on brain sections from Lgi1^+/+ and Lgi1^−/− littermate mice (aged P15). DAB peroxidase (HRP) substrate kit (Vector laboratories) was used to reveal staining.

At P15 and P30, the inner ears were extracted, and the cochleae were immediately separated and stored in cold PBS. The cochleae were perfused with 4% paraformaldehyde through the opened round and oval windows. The cochleae were fixed for 2 h and decalcified in 10% ethylenediaminetetraacetic acid solution for 1–3 h. For immunofluorescence, only the middle turn of the cochlea was kept; other tissues such as the stria vascularis, spiral ligament, and tectorial membrane were discarded.
After blocking with 10% normal goat serum in 10 mM PBS with 0.3% Triton X-100 for 1 h, the cochleae were incubated for 48 h at 4°C with primary antibodies: rabbit anti-myosin VIIa (1:800; 05012017; Proteus Biosciences, Ramona, CA, United States) for hair cells; mouse IgG1 anti-C-terminal-binding protein-2 (CtBP2; 1:500; 612044; BD Biosciences, CA, United States) for pre-synaptic ribbons in hair cells; mouse IgG2a anti-glutamate receptor 2, extracellular, clone 6C4 (GluA2; 1:2000; MAB 397; Millipore, Schwalbach, Germany) for post-synaptic glutamate receptors; and mouse anti-tubulin β3 (1:800; 801202; Biolegend, CA, United States) for SGNs and nerve fibers innervating hair cells.
The cochleae were washed three times with PBS and then incubated with appropriate Alexa Fluor-conjugated secondary antibodies (1:1000; Jackson Immuno Research, PA, United States) overnight at 4°C.