Ab133543

The cultured cells and each spinal cord slides were prepared for fluorescence microscopy by permeabilization for 5 min with 0.1% Triton- × 100, blocked with 5% BSA then incubated overnight with anti-iNOS (1:100; ab49999, Abcam Cambridge, MA, USA), anti-Arg1 (1:100; ab133543, Abcam Cambridge, MA, USA), anti-C3 (1:100; 21337–1-AP, ProteinTech, Manchester, UK), anti-S100A10 (1:100; 11250–1-AP, ProteinTech, Manchester, UK), or anti-GFAP (1:100; sc-33673, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were incubated with secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (H + L) antibody/Alexa Fluor 594 horse anti-rabbit secondary antibody (all 1:1000; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. A DAPI solution was applied for 5 min for nuclear staining. Images were captured using a Leica TCA SP8 confocal laser scanning microscopy (Leica, Germany) [26 (link)].

The macrophages in each group were collected, from which the total proteins were extracted, and their concentrations were detected through BCA method. Then, the extracted proteins were mixed well with 4×loading buffer and boiled in a metal bath at 100°C for 10 min, followed by SDS-PAGE. Next, the proteins were transferred to a PVDF membrane and incubated at 4°C overnight against the primary antibodies: IL-6 (Abcam, ab233706, 1:1000), Arginase-1 (Abcam, ab133543, 1:1000), IL-1β (Abcam, ab254360, 1:1000), tumor necrosis factor-α (TNF-α, Abcam, ab215188, 1:1000), p-phosphatidylinositol 3-kinase (p-PI3K, Abcam, ab278545, 1:1000), p-protein kinase B (p-AKT, Abcam, ab38449, 1:800), cluster of differentiation 9 (CD9, Abcam, ab236630, 1:1000), CD81 (Abcam, ab109201, 1:5000), CD63 (Abcam, ab134045, 1:5000) and GAPDH (Abcam, ab8245, 1:5000). After rinsing, they were incubated in the secondary antibody working solution of horseradish peroxidase-labeled goat anti-rabbit IgG (Abcam, ab97080, 1:5000) at room temperature for 2 hours. Subsequently, exposure and development were performed with enhanced chemiluminescent (ECL) solution, and the corresponding gray value was measured via Image J software.

Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, China). Membranes were blocked with 5% non-fat dry milk in TBST at room temperature for 2 h and incubated overnight at 4°C with primary antibodies against PPARγ (1:1000, ab59256; Abcam), inducible nitric oxide synthase (iNOS) (1:1000, ab129372; Abcam), arginase-1 (Arg-1) (1:1000, ab133543; Abcam) respectively and β-actin (1:1000, 2SGB-BIO, TA-09). Membranes were visualized with the chemiluminescence reagent, and the intensities of protein bands were evaluated by ImageJ (National Institutes of Health, United States). Protein expression was assessed relative to β-actin.