Figure 2

The expression of chOPG protein and effect on osteoclast morphology, apoptosis and resorption. 2A: Immunofluorescence assay for a possible chicken osteoprotegerin (chOPG) protein. Chicken embryo fibroblasts (CEFs) were grown on coverslips, fixed, and examined by indirect immunofluorescence. Cells were incubated with rabbit anti-chOPG serum. The secondary antibody was fluorescein-conjugated goat anti-rabbit immunoglobulin G (green). The nuclei of the corresponding cells were visualized by DAPI staining (blue). Fluorescence signals were analyzed by Fluoview microscopy (×200). Negative results are shown on card l (control group) and card 2 (pcDNA3.1 (+) transfected CEFs group), positive green fluorescence for CEFs are shown on card 3 (pcDNA3.1 (+)/chOPG transfected CEFs group). 2B: The morphology of osteoclasts was observed by inverted phase contrast microscope (×200). The adherent osteoclasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing supernatant of control group (l), pcDNA3.1 (+) transfected CEF group (2) and pcDNA3.1 (+)/chOPG transfected CEF group (3) for 5 d. 2C: Effect of the supernatant of three groups on the apoptosis of osteoclasts by flow cytometry. 2D: Toluidine blue staining of bone slices showing resorption lacunae (×200). The adherent osteoclasts were cultured in DMEM containing supernatant for 5 d in three groups.