Goat anti runx2

Four weeks after implantation, rats were sacrificed, and specimens were obtained. After fixation in 10% formalin solution for 4 days, the femurs were first decalcified in 10% ethylenediamine tetraacetic acid (EDTA) (Sigma) solution for 21 days. Using a cutting unit (Leica), 3.5 µm sections were obtained. Sections were processed for standard immunofluorescent histochemical staining and analysis by using primary antibodies, goat anti‐Runx2 (an endothelial cell marker; R&D Systems) and rabbit anti‐OCN (an osteoprogenitor marker; Abcam, Cambridge, England), as well as Alex 594‐conjugated secondary antibodies to corresponding species (Millipore, Billerica, MA). Cell nuclei were stained with 40, 60‐diamidino‐2‐phenylindole (DAPI, Sigma). After being air dried and coverslipped, the sections were observed under a confocal laser scanning microscope (FV‐1000, Olympus, Tokyo, Japan) with the appropriate laser beams and filter settings, and confocal images were captured. For quantitative analysis of the osteoblasts around the implants, a ROI was defined as a ring around the screw extending 200 µm from the lowest point of the thread grooves and barring the region of the screw threads. The area ratio of the total number of Runx2+ and OCN in the ROI was determined with ImageJ software. Four nonadjacent slices of each sample were analysed, with five samples in each group.