Mouse anti nr2b

Brains were fixed with 4% paraformaldehyde, dehydrated in 30% sucrose and stained using the Vectastain system (Vector Labs, Burlingame, CA, USA), as described previously.^{57 (link)} Immunostaining with mouse anti-NR2B (1:2000; Abcam, Cambridge, MA, USA), followed by a secondary goat anti-mouse antibody and avidin–biotin complex, was visualized with diaminobenzidine. Sections were observed with a light microscope (Leica, Buffalo Grove, IL, USA) to verify the expression of NR2B in RSC.

Rats were anesthetized and perfused through the ascending aorta with NS, followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer. Lumbar enlargement segments were then harvested, postfixed at 4 °C overnight, and dehydrated in 30% sucrose. Afterwards, 30-μm free-floating transverse sections were cut at −20 °C in a freezing microtome. Sections were collected in phosphate-buffered saline and double-labeled to investigate the co-expression of NR2B and neuronal, glial, or microglial markers. Specifically, spinal sections were incubated for 48 h at 4 °C in a mixture of mouse anti-NR2B (1:200; Abcam) and rabbit anti-PSD-95 (1:2000; Abcam), rabbit anti-NeuN (1:500; Abcam), rabbit anti-GFAP (1:500; Proteintech, IL), or rabbit anti-Iba-1 (1:1000; Abcam) antibodies. The sections were then incubated with a mixture of Cy3-conjugated anti-mouse IgG (1:200; Proteintech) and fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200; Proteintech), for 2 h at room temperature. Fluorescent signal was viewed with a fluorescence microscope (OLYMPUS, x-Cite 120, Japan) with the appropriate filters.

Protein concentration was determined by the BCA protein assay kit. Proteins (20 μg) were resolved in SDS-polyacrylamide gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Nonspecific binding sites on the blotting membrane were blocked with 5% non-fat dry milk for 2 h and incubated at 4°C overnight with mouse anti-NR2B (1∶1000, Abcam), rabbit anti-SAP102 (1∶500, Santa Cruz), rabbit anti-CaMKII (1∶1000, Cell Signaling), rabbit anti-SynGAP (1∶1000, Abcam), rabbit anti-pERK1/2 (1∶2000, Cell Signaling), rabbit anti-pCREB (1∶1000, Cell Signaling Technology) and rabbit anti-BDNF (1∶1000, Sigma). The proteins were detected with anti-rabbit or anti-mouse secondary antibody for 2 h at room temperature. Then, the proteins were visualized with BeyoECL Plus reagents and captured by an enhanced chemiluminescence system (ECL kit, Pierce Biotechnology, Inc.).