Dab color developing solution

After the micro-CT imaging and analysis were performed, the excised femora were decalcified using 10% EDTA (pH = 7.4) at room temperature for 2 weeks. The samples were dehydrated using a gradient ethanol series and a final xylene step and were subsequently paraffin embedded. Approximately 5-μm-thick sections were made. Sectioning of the paraffin-embedded samples was performed along the longitudinal axis of the femur. Longitudinal sections were prepared using a microtome (Leica, USA) and tungsten carbide blades. Sections were stained with hematoxylin–eosin and saffran (Beyotime, China). Images of the femur defect region were acquired via light microscopy. For immunohistochemical analysis, bone sections were incubated with primary antibodies against anti-phospho Smad1/5/8 (Millipore, USA) overnight at 4 °C, and then covered with secondary antibodies (Servicebio, China) at room temperature for 50 min. After the sections were cleaned in PBS, DAB color developing solution (Servicebio, China) was used for color development. The slices were flushed with tap water to terminate color development. The slices were stained with hematoxylin for approximately 3 min and rinsed with water. Finally, dehydrated seal was performed, and the images were collected and analyzed with a microscope.