Rabbit anti nr2b

After beam walking test, mice were sacrificed by pentobarbital overdose and received intracardiac perfusion with ice-cold PBS and paraformaldehyde. Cervical spinal cords were harvested, and sectioned at 10-μm thickness with a cryostat (Thermo Fisher Scientific, USA). Immunofluorescence was performed with standard procedure. The following primary antibodies were used: (1) rabbit anti-human IgG (1:600, Dako, USA), (2) rabbit anti-AQP4 (1:200, Sigma-Aldrich, USA), (3) mouse anti-glial fibrillary acidic protein (GFAP, 1:200, Santa Cruz Biotechnology, USA), (4) goat anti-myelin basic protein (MBP, 1:200, Dako, USA), (5) rabbit anti-neurofilament heavy polypeptide (NF-H, 1:400, Sigma-Aldrich, USA), (6) rabbit anti-NR2B (1:200, Abcam, UK), (7) rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1, 1:200, Wako, Japan), (8) rat anti-lymphocyte antigen 6 complex locus G6D (Ly6G, 1:400, Abcam, UK), (9) rat anti-CD4 (1:100, Santa Cruz Biotechnology, USA), and (10) rat anti-CD8 (1:100, Santa Cruz Biotechnology, USA). Sections were then incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies (Thermo Fisher Scientific, USA) at room temperature for 1 h. They were counterstained with DAPI and mounted with anti-fade reagent (Thermo Fisher Scientific, USA).

Rats were deeply anesthetized using pentobarbital sodium and decapitated, and then the brain was harvested, and the ACC was divided into left and right sides for protein analysis, as described in our previous publication^{10 (link)}. The protein samples were separated by SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis) and transferred to a polyvinylidene difluoride membrane. In most experiments, membranes were cut according to protein marker and blocked in 5% skimmed milk at room temperature for 1 h. Then, the fragments were incubated with the corresponding primary antibodies for 24 h at 4 °C: rabbit-anti-NR2B (1:500, Abcam, Cambridge, UK), rabbit-anti-PSD-95 (1:2000, Cell Signaling Technology, Danvers, MA, USA), rabbit-anti-CREB (1:1000, Cell Signaling Technology), rabbit-anti-pCREB (1:1000, Cell Signaling Technology), and mouse-anti-GAPDH (1:10,000, Proteintech, Rosemont, IL, USA). The membrane was incubated in horseradish peroxidase-conjugated secondary antibody for 2 h at 23 °C room temperature after TBST washing. The ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) was used to detect immune complex bands.

The perilesional cortex was collected under a dissecting microscope at 7 days after TBI and extracted in RIPA lysis buffer containing 1% (v/v) protease and phosphatase inhibitor cocktail. The protein samples were separated by SDS-PAGE and transferred to PVDF. After being blocked with 5% (w/v) non-fat milk, the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-Cleaved caspase 3 (1:1,000, Cell Signaling Technology, United States), Rabbit anti-DAPK1 (1:1,000, Cell Signaling Technology, Massachusetts, MA, United States), Rabbit anti-β-actin (1:1,000, Abcam, Cambridge, United Kingdom), Rabbit anti-NR2B (1:1,000, Abcam, Cambridge, United Kingdom), Rabbit anti-p-NR2B (Ser1303, 1:1,000, Abcam, Cambridge, United Kingdom), Rabbit anti-PSD95 (1:1,000, Proteintech, China), Rabbit anti-ERK1/2 (1:1,000, Cell Signaling Technology, Massachusetts, MA, United States), Rabbit anti-p-ERK1/2 (Thr202/Tyr204, 1:1,000, Cell Signaling Technology, Massachusetts, MA, United States). Afterward, the membranes were incubated with HRP anti-rabbit (1:1,000, Cell Signaling Technology, Massachusetts, MA, United States) for 1 h at room temperature and then scanned with a Bio-Rad (California, United States) gel imaging system.