Histology and immunohistochemistry

After bone defect treatment, the rat femurs of the affected side were removed and placed into 4% PFA solution for 24-h immobilization. Then, the femurs were dehydrated and decalcified with equal-concentration gradient ethanol, embedded in paraffin, and cut into 5-um sections with a rotary microtome. After being deparaffinized, we performed hematoxylin-eosin (HE) staining and Masson trichrome staining to assess bone defect healing and formation of bone collagen. In addition, immunohistochemical staining was performed to observe the expression of vascular endothelial growth factor receptor 2 (VEGFR2) and immunofluorescence staining was used to assess vascular growth status.