Ab210823

For IF staining, a sterile round glass slide (18 mm) was placed on a 12-well plate and coated with 1× poly-lysine overnight. BV2 cells were seeded into the 12-well plate at a concentration of 1 × 10⁵/cells the next day. Following the intervention, the treated cells were fixed for 20 min in precooled 4% PFA and permeabilized for 20 min at room temperature with 0.1% Triton X-100 (#T8200 Solarbio, Beijing, China). The fixed cells were blocked for 30 min at 37 °C with 3% bovine serum albumin (A6020 Biotopped, Beijing, China) and the sections were incubated overnight at 4 °C with primary antibodies against Iba-1 (1:800 #17198 Cell signaling, Danvers, MA, USA), iNOS (1:500 ab210823, Abcam, Cambridge, UK), and Arg-1(1:200 ab239731, Abcam, Cambridge, UK). Then, the sections were rinsed three times with PBS for 5 min before incubation for 1 h at room temperature with Alexa Fluor 488 goat anti-rabbit IgG (1:500, a-11008, Thermo Fisher Science, Waltham, MA, USA) and Alexa Fluor 555 donkey anti-mouse IgG (1:500, a-31570, Thermo Fisher science, MA, USA). Prior to the addition of DAPI, the sections were washed three times with PBS and then sealed with a coverslip. A fluorescence microscope (Olympus BX61, Tokyo, Japan) was used to observe the signals. The IF images were analyzed using ImageJ software (version 1.53, National Institutes of Health, USA).

Mouse-derived macrophage Raw264.7 was purchased from Shanghai Yuanzhixin Biotechnology Co (Shanghai, China). Mouse bone marrow-derived macrophages were primarily extracted from wild-type Balb/c mice, and the methods have been detailed previously [5 (link)]. Mouse aortic endothelial cells (MAECs) were purchased from Otwo Biotech Co (Shenzhen, China). CD68 antibodies (ab125212), inducible nitric oxide synthase (iNOS) antibodies (ab210823), CD31 antibodies (ab24590), α-SMA antibodies (ab21027), Collagen-I antibodies (ab260043), VE-Cadherin antibodies (ab33168), GADPH antibodies (ab9485), Alexa Fluor® 488-labeled donkey anti-mouse secondary antibody (ab150109), Alexa Fluor® 555-labeled donkey anti-rabbit secondary antibody (ab150062) were purchased from Abcam Co (Cambridge, MA, USA); Vimentin antibodies (10366-1-AP), F4/80 antibodies (28463-1-AP), CD206 antibodies (60143-1-Ig), Interferon-γ (IFN-γ) were purchased from Proteintech Co (Chicago, IL, USA); CD206 Cell Flow antibodies (17-2061-80), lipopolysaccharide (LPS; 00⁃4976⁃93) were purchased from Thermo Fisher Scientific Co (Waltham, MA, USA); iNOS Flow Cytology antibodies (130-116-421) was purchased from Miltenyi Biotec Co (Bergisch Gladbach, Germany); The primers were designed and synthesized by Nanjing DynaScience Biotechnology Co. TNF-α Antagonist III (R-7050; 303997-35-5) was purchased from Selleck. Co (Shanghai, China).

After 7 days of SCI, the rats were perfused with paraformaldehyde, dissected, and sectioned at the site of injury, paraformaldehyde-fixed, sucrose sunk and OCT embedded, followed by sectioning. Sections were treated with 0.3% TritonX-100 for 20 min and blocked with 5% BSA blocking solution at room temperature for 1 h. The appropriate primary antibody was added to the sections and incubated overnight at 4°C, including anti-FECR1G (1:100, DF13263, Affbiotech, China), anti-NFATC2 (1:100, A3107, Abclonal, China), and anti-iNOS (1:100, ab210823, Abcam, United Kingdom). Sections were incubated with a secondary antibody (1:200) at room temperature in the dark for 1 h. After washing with PBS for 10 min, cell nuclei were stained with DAPI.

Paraffin-embedded sections of the mouse aorta were taken, dewaxed, dehydrated, and subjected to antigen retrieval. Sections were added with 0.03% Triton for 10 min and sealed with normal goat serum blocking solution (C-0005, HaoranBio, Shanghai, China) for 60 min at room temperature. For macrophages, macrophages were fixed with 4% paraformaldehyde for 30 min, washed with precooled PBS And sealed as the above. Next, primary antibodies of HMGB1 (66525-1-Ig, 1:250, Mouse, Proteintech, VA, USA), F4/80 (ab6640, 1:50, Rabbit, Abcam), GSDMD (sc-393581, 1:200, Mouse, SANTA, UC, USA), iNOS (ab210823, 1: 100, Mouse, Abcam), and CD206 (60143-1-Ig, 1:200, Mouse, Proteintech) were selected for sample incubation at 4 ℃ overnight. Then, the samples were incubated with fluorescent secondary antibody, Alexa Fluor^® 647-Anti-Rabbit IgG (ab150075, 1:500, Donkey, Abcam) or Alexa Fluor^® 488-Anti-Mouse IgG (ab150113, 1:500, Goat, Abcam) at room temperature for 60 min in the dark. A fluorescence microscope was utilized for observation, with fluorescence intensity recorded.

For immunofluorescence staining, brain sections were pretreated with citrate buffer for 5 min for antigen retrieval, then blocked with immunostaining blocking solution (P0102, Beyotime, China) at room temperature for 1 h. Then the sections were incubated with rabbit anti-Oligo2 (1:100, ab109186, Abcam, UK) overnight at 4 °C. For double immunofluorescence staining, the sections were incubated with mixtures of rabbit anti-IBA-1 (1:300, 019-19741, Wako, Japan) or mouse anti-IBA-1 (1:300, 016-26721, Wako, Japan) and rabbit anti-TMEM119 (1:100, NBP2-30551, Novus, USA), mouse anti-iNOS (1:100, ab210823, Abcam, UK), rabbit anti-CD206 (1:100, ab64693, Abcam, UK), rabbit anti-TREM2 (1:200, PA5-87933, ThermoFisher, USA), and mouse anti-activin-A (1:50, ab89307, Abcam, UK); and mixtures of rabbit anti-NG2 (1:100, AB5320, Millipore, USA) and mouse anti-activin-A (1:50, ab89307, Abcam, UK), Acvr2B (1:100, ab128544 and ab180185, Abcam, UK); The next day, sections were incubated with secondary antibodies after washing with phosphate-buffered solution (PBS). In addition, sections were incubated with Rabbit anti-BCAS1 FITC conjugated (1:50, C01226F, SAB, China). Finally, sections were mounted with a solution containing 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were observed under a microscope (BX63, Olympus).