Immunofluorescent microscope

mMCs were collected and concentrated on a microscopy slide by cytospin centrifuge. Cells were air-dried at room temperature and fixation/permeabilization solution kits (BD biosciences, San Jose, CA, USA) were used before staining. Purified anti-Lcn2 mouse antibody, clone: M0410B2 (Biolegend, San Diego, CA, USA), was used for primary staining overnight. Alexa Fluor 488 goat anti mouse IgG secondary antibodies (Thermo Fisher, Waltham, MA, USA) were applied for fluorescence conjugation. Incubation with secondary antibodies was only used as a negative control (Figure S2). Slides were mounted in ProLong Anti-Fade reagent with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes). Slide images were captured by an immunofluorescent microscope (Olympus, Center Valley, PA, USA)

Rat glioma C6 cell lines provided by American Type Culture Collection (ATCC) were grown in RPMI medium 1640 (Life Technologies) containing 4% fetal bovine serum and 8% horse serum at 37°C in a 5% CO₂ incubator. Three groups were designated as follows: the first was control, the second was pre-treatment with rat TNF-α (10 pg/mL; R&D Systems) for 1 h, and the third was pre-treatment with rat TNF-α for 1 h with etanercept (0.1 μg/ml) added for 30 min. C6 cells were seeded (1 × 10⁶) onto glass slides overnight and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 15 min. The cells were then rinsed twice with PBS and permeabilized with 1% Triton X-100 for 7 min. Next, the cells were pretreated with 1% bovine serum albumin (BSA) in PBS at 25°C for 60 min, incubated with rabbit anti-ADMA polyclonal antibodies (Millipore) at a dilution of 1:1000 for 1 h, and treated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG polyclonal antibodies at a dilution of 1:10,000 for 1 h. Finally, the cells were washed with PBS, mounted in 90% glycerol containing 4′,6-diamidino-2-phenylindole (DAPI), and photographed under an immunofluorescent microscope (Olympus). In addition, the concentrations of ADMA in the whole cell lysate were examined by ELISA.

After blocking with 3% bovine serum albumin (BSA), cells on the coverslip were stained with phalloidin 568 (Invitrogen) to detect F-actin at room temperature for 1 h. Stained coverslips were mounted with 90% glycerol containing DAPI (Invitrogen), and photographed under the immunofluorescent microscope (Olympus).