Mab240 100

Western blot was performed based on the manufacturer’s specification. Briefly, isolated brain tissues (100 mg) or primary cultured cells (5 × 10⁶) were resuspended in RIPA lysis buffer (Beyotime) with PMSF. Lysed protein was quantified by BCA protein assay (BCA, Beyotime), separated by SDS-PAGE (10%) and transferred toward PVDF membranes. PVDF blots were then incubated with primary antibodies recognizing rabbit anti-TRPV1 [(1:1,000, NB100-98886, Novus), goat anti-Iba1 (1:500, NB100-1028, Novus), mouse anti-CD68/ED1 (1:1,000, ab31630, Abcam), mouse anti-TLR4 (1:1,000, sc-293072, Santa Cruz, CA, USA), mouse anti-TGF-β1 (1:1,000, MAB240-100, R&D), rat anti-TβR I (1:1,000, MAB5871, R&D), goat anti-TβR II (1:1,000, AF532, R&D), rabbit anti-Arg1 (1:1,000, 9819, CST), goat anti-Ym1 (1:1,000, AF2446, R&D), rabbit anti-GAPDH (1:1,000, ab9485, Abcam), or mouse anti-β-actin (1:100,000, 60008, proteintech)]. After incubation with the corresponding HRP-conjugated secondary antibody, immunoreactive bands were developed by enhanced chemiluminescence (ECL) detection reagent.

For immunohistochemical analysis, thin sections blocked by 5% goat serum followed by incubating in specific primary antibodies. PBS was applied to wash the sections three times. Then, samples was stained with horseradish peroxidase-conjugated secondary antibodies and visualized by substrate DAB. Images were taken with a microscope (Leica, 200 ×) under same acquisition settings for each section. The primary antibodies were used as follow: TGF-β (MAB240-100, R&D System, 1:600 dilution), F4/80 (LS-C96373-100, Lifespan, 1:1000 dilution), CD68 (ab125212, Abcam, 1:600 dilution), IL-1β (SRP8033, Sigma, 1:1000 dilution), TNF-α (ab6671, Abcam, 1: 600 dilution).