Cd206

Animals from each treatment group were euthanized at 8 weeks, and the tibiae were extracted. Samples were decalcified and embedded in paraffin and 5 μm sections were prepared. Prior to staining, sections were deparaffinized and antigen retrieval was performed at 95 °C with 10 mM citrate buffer for 8 min. Sodium borohydride solution (100 mg/mL) was used to reduce background autofluorescence of the tissue. Samples were permeabilized with 0.2% Triton X-100 and then blocked using 10% donkey and goat serum to prevent non-specific antibody binding. Immunofluorescence staining was performed using anti-GFP (Abcam), anti-Osteocalcin (Abcam), anti-alpha Smooth Muscle Actin (Abcam), isolectin IB4 (Life Technologies), anti-CCR7 (Abcam), CD206 (Bioss), or CD90 (BioLegend). Samples were incubated with primary antibody overnight at 4 °C, and secondary antibody for one hour at room temperature. Slides were mounted in 100 mM glycine and images were taken on the Zeiss LSM 700 confocal microscope using Zen software (Zeiss). Quantification of immunohistochemistry was performed by counting the number of cells positive for the specified markers by identifying a threshold based on secondary only controls. For identification of newly formed bone, sections were stained with hematoxylin and eosin and then imaged for autofluorescence using an upright microscope.