Anti mouse cd206

Rats were anesthetized with 2% sodium pentobarbital in saline (60 mg/kg, i.p.; Sigma, St Louise, MO, USA) and perfused with saline and then perfused with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) on day 14 after surgery. Brains were collected and fixed in the 4% PFA for 2 h and dehydrated in 30% sucrose at 4°C overnight. 10-mm-thick coronally sections of the prefrontal cortex were cut and pasted on glass slides. After blocking with 10% norm goat serum for 1 h at room temperature, the slices were incubated with primary antibodies: rabbit anti-CD11b (1:250; Abcam, Cambridge, UK), rabbit anti-Iba1 (1:500; Wako, Japan), mouse anti-CD206 (1:500; Biolegend, San Diego, USA), or mouse anti-8-hydroxy-20-deoxyguanosine (8-OH-dG; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) in 1% BSA at 4°C overnight. After washing with PBS for 3 × 5 min, the slices were exposed to the secondary antibodies Alexa fluor 488/549 goat anti-rabbit and Alexa fluor 488/549 goat anti-mouse (1:400; Bioworld Technology, St. Louis Park, MN, USA), and DAPI (1:1,000; Sigma, St. Louis, MO, USA) for 1 h at room temperature. A confocal microscope (Leica, TCS SP2, Germany) was used to capture the fluorescent images. The immunofluorescence intensity was calculated by Image J (Wayne Rasband, National Institute of Health, USA).

Calvarial of mice were harvested at day 3, week 2 and week 8 for histology. Tissues were fixed overnight at 4% paraformaldehyde, demineralized for 2 weeks in 16% ethylenediaminetetraacetic acid, and embedded in OCT for cryo-sectioning. Tissue morphology was examined by H&E staining (Sigma, St. Louis, MO, USA) and Masson trichrome staining (Thermo Scientific, Waltham, MA) according to manufacturer's instructions. For immunostaining, 20μm sectioned slices were treated with blocking buffer consisting of 10% bovine serum albumin in 1X PBS and incubated with rat anti CD31 (BD Biosciences, San Jose, CA, USA, 1:50 dilution), rabbit anti luciferase (abcam, Cambridge, MA, USA, 1:50 dilution), mouse anti iNOS (BD Biosciences, 1:50 dilution) and mouse anti CD206 (Biolegend, San Diego, CA, USA, 1:50 dilution) overnight at 4 ℃. After washing in PBS for 3 times, sectioned slices were incubated for 1 h at room temperature with secondary antibodies. Nuclei was counterstained with Hoechst 33342 stain (Thermo Scientific) and images were taken under Zeiss fluorescence microscope. Sections were stained with all reagents without primary antibody for negative controls.