2.11 Western blot
Western blot was routinely performed as previously reported [32]. Briefly, brain tissues were quickly dissected out on ice and resolved in RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail and phosphatase inhibitor (Beyotime, Shanghai, China). Denatured proteins in SDS-loading buffer were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, United States). After blocking, the membranes were incubated overnight at 4 ◦C with primary antibodies involving anti-TNF-α (Santa Cruz), anti-iNOS (Proteintech), anti-Arg1 (Santa Cruz), anti-TRPM4 (Sigma-Aldrich) and anti-β-actin (Proteintech). After washing with TBST, bands incubated with HRP-conjugated secondary antibodies (Proteintech) were detected by enhanced chemiluminescence advance Western blotting detection reagents (FDbio, Hangzhou, China). The intensities of protein bands were quantified and normalized to the level of β-actin by using ImageJ software.