The calvariae were decalcified in 10% EDTA solution, embedded in paraffin, and sectioned into 5- to 10-μm sections. Hematoxylin and eosin (H&E) staining was performed as per previously published protocols (Huang et al., 2020 (link)).
For immunofluorescent staining, the slides were pre-treated with 5% BSA blocking buffer for 1h at room temperature and stained for osteomarkers and macrophage-specific antigens using mouse monoclonal [65529.111] anti-bone morphogenetic protein 2 (BMP2) antibody (1/100, ab6285, Abcam), mouse monoclonal [OCG3] anti-osteocalcin (OCN) antibody(1/100, ab13420, Abcam), rabbit monoclonal anti-iNOS antibody (1/100, ab15323, Abcam), rabbit monoclonal anti-CD206 antibody (1/100, ab64693, Abcam), and mouse monoclonal [3A6] anti-IL-1β antibody (1/100, 12242, Cell Signaling). Sections were then stained with anti-mouse FITC and anti-rabbit TRITC secondary antibodies (1/200, Sigma), imaged using Zeiss LSM 710 laser scanning confocal microscope equipped with Zen image analysis software. For a vascular marker, sections were stained with rabbit polyclonal anti-CD31 antibody (1/100, ab28364, Abcam) and peroxidase-conjugated secondary antibody. ImageJ was used to measure positive immunostained cell number or % area per field (n = 4 per group). The positive cell number of iNOS and CD206 was divided by the number of nuclei per field.
For immunofluorescent staining, the slides were pre-treated with 5% BSA blocking buffer for 1h at room temperature and stained for osteomarkers and macrophage-specific antigens using mouse monoclonal [65529.111] anti-bone morphogenetic protein 2 (BMP2) antibody (1/100, ab6285, Abcam), mouse monoclonal [OCG3] anti-osteocalcin (OCN) antibody(1/100, ab13420, Abcam), rabbit monoclonal anti-iNOS antibody (1/100, ab15323, Abcam), rabbit monoclonal anti-CD206 antibody (1/100, ab64693, Abcam), and mouse monoclonal [3A6] anti-IL-1β antibody (1/100, 12242, Cell Signaling). Sections were then stained with anti-mouse FITC and anti-rabbit TRITC secondary antibodies (1/200, Sigma), imaged using Zeiss LSM 710 laser scanning confocal microscope equipped with Zen image analysis software. For a vascular marker, sections were stained with rabbit polyclonal anti-CD31 antibody (1/100, ab28364, Abcam) and peroxidase-conjugated secondary antibody. ImageJ was used to measure positive immunostained cell number or % area per field (n = 4 per group). The positive cell number of iNOS and CD206 was divided by the number of nuclei per field.
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