To confirm the RANK receptor targeting potential of generated mAb in vitro, osteoclast-like cells were generated in Lab Tek II chamber slide system (Nunc) from RAW 264.7 cells, as described below for generation of osteoclasts. The MG-63 osteoblast-like cell line known to lack RANK receptor (16) was used as negative control. Likewise, osteoclast cell culture omitting primary mAb was also used as a method negative control. Cell cultures were washed with PBS, fixed in 4% paraformaldehyde in PBS (pH 7.4) for 5 min and rinsed thoroughly. After blocking with 3% BSA in PBS for 1 h, the cells were incubated with our anti-RANK mAb (4 μg/ml) overnight at 4°C. Antibody detection was carried out using rabbit anti-mouse IgG-FITC (1:100; Sigma, USA) for 60 min at 4°C. To visualize cell nuclei, slides were counterstained with 1.5 μg/ml 4-6diamidino-2 phenylindole (DAPI, Sigma) diluted in PBS. Culture slides were separated from their chambers, mounted and photographed using confocal microscopy (Zeiss LSM 710 with ZEN software and the microscope the Observer.Z1).