Ab283319

The L4–L5 spinal cord tissues of three rats in each group were selected and fixed in 10% neutral formalin, dehydrated in ethanol, embedded in wax, and then sectioned at a thickness of 4 µm. Spinal cord sections were dewaxed and incubated overnight at 4 °C with CD80 antibody (1:100, bs-2211R, Bioss, Beijing, China) and Iba-1 antibody (1:100, ab283319, Abcam, Cambridge, UK); CD206 antibody (1:100, DF4149, Affinity, Suzhou, China) and Iba-1 antibody (1:100, ab283319, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C; C3 antibody (1:100, DF13224, Affinity, Suzhou, China) and GFAP antibody (1:100, ab279290, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C; S100A10 antibody (1:100, bs-8503, Bioss, Beijing, China) and GFAP antibody (1:100, ab279290, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C. Goat anti-rabbit IgG H&L (Alexa Flour 488,1:200, ab150077, Abcam, Cambridge, UK) and goat anti-mouse IgG H&L (Alexa Flour 647,1:200, ab150115, Abcam, Cambridge, UK) were used as secondary antibodies. Tissue sections were immunofluorescently stained and then observed under a laser confocal microscope (SP8, Leica, Wetzlar, Germany) and photographed. Fluorescence quantitative analysis was performed by Image J software.

Rat L4-L6 spinal cord samples were fixed, dehydrated, and frozen in a -80 °C freezer, and cryosectioned at 8 μm thickness for subsequent studies. The removed sections were dried for 10 min and rinsed thrice with phosphate-buffered saline (PBS) for 10 min each. Sections were rinsed thrice for 10 min each after breaking the membranes with 0.5% Triton for 2 h. Following repair using citrate solution, sections were naturally cooled and then rinsed thrice for 10 min each. Subsequently, sections was blocked with 2% bovine serum albumin and incubated overnight with following primary antibodies: anti-ionized calcium-binding adaptor molecule-1 (Iba-1) (red; 1:100, ab283319, Abcam, Cambridge, UK), anti-DNMT1 (green; 1:250, ab188453, Abcam), anti-iNOS (green; 1:250, ab178945, ab283319, Abcam), anti-Arg-1 (green; 1:200, ab96183, Abcam) at 4 °C, followed by washing with 0.01 moL/L PBS. Afterwards, sections were reacted for 1 h with secondary antibody immunoglobulin G (IgG, 1:2000, ab205718, Abcam) at 37 °C under dark conditions, and then washed. Following staining the nuclei with Hoechst for 10 min, sections were rinsed five times for 10 min each. The expression and localization of DNMT1, iNOS, and Arg-1 in microglia were determined after mounting using glycerol, with Iba-1 as a marker of microglia.

The mice were deeply anesthetized with sodium pentobarbital via intraperitoneal injection at a dose of 60 mg/kg. Afterwards, the mice were perfused with cold PBS for 5 min, followed by 4% paraformaldehyde solution for 15 min. Then, the brains were excised and postfixed overnight in 4% paraformaldehyde at 4 ℃ and dehydrated in 30% sucrose–PBS. The brains were mounted in optimal cutting temperature embedding medium, frozen and cut coronally at a 40 μm thickness on a cryostat. Next, the brain slices were fixed with 4% paraformaldehyde for another 10 min and incubated with 0.3% Triton X-100 for 10 min, followed by blocking with 10% normal donkey serum containing 0.1% Triton X-100 for 60 min at room temperature. Afterwards, the brain sections were incubated overnight at 4 ℃ with the following primary antibodies: mouse anti-NeuN monoclonal antibody (Proteintech, 66836-1-Ig), mouse anti-Iba1 monoclonal antibody (Abcam, ab283319) and rabbit anti-Smad7 polyclonal antibody (Proteintech, 25840-1-AP). After being washed three times in PBS, the brain sections were incubated with secondary antibodies conjugated with Alexa 488 or 555 for 60 min at room temperature. The slices were mounted with Antifade Mounting Medium containing DAPI (Beyotime, China) and observed using a laser scanning confocal microscope (Leica CM DMI8).