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.