Fluoroshield with dapi

To collect the spinal cords after contusion injury or sham operation mice, mice were anesthetized and then rapidly perfused transcardially with 0.9% saline, followed by 4% PFA. Then, the spinal cords were cryoprotected in 30% sucrose for 3 d at 4°C, before being sectioned. Serial cryostat sections (14 mm thick) were obtained. For immunofluorescent staining, the sectioned slices were permeabilized with 0.3% Triton X-100 in PBS for 30 min and then blocked with 5% BSA for 1 h. After incubation with goat anti-CD31 Alexa Fluor 488-conjugated antibody (1 : 100, R&D, USA), rabbit anti-ki67 (1 : 200, Invitrogen, USA), and rabbit anti-Nrf2 (1 : 200, Proteintech) overnight at 4°C, the sectioned slices were incubated with secondary antibodies anti-rabbit Alexa Fluor 488 or Alexa Fluor 568 (1 : 400, Abcam) for 1.5 h. The slices were mounted with Fluoroshield™ with DAPI (GeneTex Inc.). Signals were analyzed by a fluorescence microscope (Zeiss Apotome 2). Positive cells were quantified using the ImageJ software.
Cell samples were fixed with 4% PFA for 20 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with 5% BSA for 30 min and then incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation for 1.5 h. After washing with PBS, cells were stained with DAPI.

Cells were fixed in cold methanol for 2 min. After permeation with 0.25% Triton X-100 for 1 hour, the cells were blocked with 5% bovine serum albumin for 2 hours. The cells were incubated with primary antibodies (1:200 dilution) for 24 hours and then with secondary antibodies (1:500 dilution) for 2 hours. The secondary antibodies donkey anti-rabbit immunoglobulin G (IgG)–Alexa Fluor 568 and goat anti-mouse IgG–Alexa Fluor 488 were purchased from Abcam and Life Technologies, respectively. The cover slides were mounted in Fluoroshield with DAPI (GeneTex) and analyzed using a Leica TCS SP5 confocal microscope.

The HSC3 cells were plated on to coverslips at a density of 5 × 10⁴/24 well/coverslip. After three washes with PBS, they were treated with or without FW for 30 s. The cells were further incubated with 10% FCS-RPMI for 3 h following which, they were fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the permeabilization of the cells was performed by incubating them with 1% Triton X-100 solution (1% Triton X-100 in PBS) for 30 min. The cells were incubated with 1% BSA-PBS for 1 h to block the non-specific reaction; subsequently, they were incubated with rabbit anti-human IL-1α Ab (Abcam; ×100 dilution with 1% BSA-PBS) for another 18 h. After three wash with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG for 18 h. The cells were extensively washed with PBS and mounted on glass slides using Fluoroshield with DAPI (Gene Tex, California, USA). Images were taken using a fluorescence microscope (All-in-one Fluorescence Microscope, Keyence, Osaka, Japan).