Fractured tibias were fixed in 4% PFA, decalcified in 10% EDTA for 3 weeks, and processed for paraffin embedding. A series of 6-μm-thick longitudinal sections were cut across the entire fracture callus from one side of cortical bone to the other. For each bone, a central section with the largest callus area, as well as two sections at 192 μm (~¼ bone width) before and after the central section were stained with Safranin-O/Fast green and quantified for cartilage area, bone area, and fibrosis area by ImageJ. Paraffin sections adjacent to the central sections were used for picrosirius red staining and IHC. After antigen retrieval, slides were incubated with primary antibodies, including rabbit anti-osteocalcin (Takara Clontech, Mountain View, CA, USA; m173), rabbit anti-VEGF (Abcam, Cambridge, MA, USA; AB46154), goat anti-Osterix (Santa Cruz Biotechnology, Dallas, TX, USA; sc-22538), rabbit anti-type II Collagen (Abcam, AB34712), goat anti-Sox9 (R&D Systems, Minneapolis, MN, USA; AF3075), and rat anti-Endomucin (Santa Cruz Biotechnology, sc-65495), at 4°C overnight, followed by binding with biotinylated secondary antibodies and DAB color development. For TRAP staining, tartrate-resistant acid phosphatase (TRAP) assay kit (Sigma-Aldrich, St. Louis, MO, USA; 387A-1KT) was used.
To obtain frozen sections for immunofluorescent imaging, fractured tibias were fixed in 4% PFA for 1 day, transferred to 30% sucrose in PBS overnight, and embedded in OCT compound (ThermoFisher Scientific, Waltham, MA, USA) for frozen sectioning at a thickness of 6 μm, aided by the use of cryofilm 2C (SECTION-LAB Co. Ltd., Hiroshima, Japan). After glued to slides, sections were incubated with rat anti-Endomucin at 4°-C overnight followed by Alexa Fluor 488-conjugated goat anti-Rat IgG secondary antibody (Abcam, ab150157). For EdU staining, mice received 1.6 mg/kg EdU 3 hours before death and the staining was carried out according to the manufacturer’s instructions (Click-iT™ EdU Alexa Fluor™ 647 Imaging Kit, ThermoFisher Scientific, c10340).
To obtain frozen sections for immunofluorescent imaging, fractured tibias were fixed in 4% PFA for 1 day, transferred to 30% sucrose in PBS overnight, and embedded in OCT compound (ThermoFisher Scientific, Waltham, MA, USA) for frozen sectioning at a thickness of 6 μm, aided by the use of cryofilm 2C (SECTION-LAB Co. Ltd., Hiroshima, Japan). After glued to slides, sections were incubated with rat anti-Endomucin at 4°-C overnight followed by Alexa Fluor 488-conjugated goat anti-Rat IgG secondary antibody (Abcam, ab150157). For EdU staining, mice received 1.6 mg/kg EdU 3 hours before death and the staining was carried out according to the manufacturer’s instructions (Click-iT™ EdU Alexa Fluor™ 647 Imaging Kit, ThermoFisher Scientific, c10340).
