Ix81 confocal microscopy systems

Cultured cells were fixed with 2% formalin (Sigma) for 20 min at room temperature (RT), blocked with 1% BSA for 30 min, stained sequentially with primary antibodies against pericentrin (Abcam) and tubulin (Sigma) for 1 h at RT, and then incubated with secondary antibodies conjugated to Alexa-488 or −680 for 30 min. The nuclei were stained for 15 minutes with PBS containing 1 μg/ml of 4, 6-diamidino-2-phenylindole (DAPI, Sigma) before mounted with the anti-fade agent Prolong (Molecular Probes). Confocal analyses were performed with Olympus IX81 confocal microscopy systems.

For 3D cultures assay, cells were cultured according to manufacturer's instructions. Briefly, 1,000 single cells per well were plated in an eight-well chamber slide (BD Biosciences) coated with Matrigel from BD Biosciences. Cells were grown in assay medium with DMSO or 1 μM of MGCD. Medium was changed every 4 days. For 3D immunofluorescence, the cell spheroids were fixed with 2% formalin (Sigma) for 20 min at RT, permeabilized with PBS containing 0.5% Triton X-100 for 10 min at 4°C, and blocked in blocking Buffer (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 7.7 mM NaN₃, 0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20, 10% goat serum) for 45-60 min at RT, and incubated with primary antibody against Ki67 (BD Biosciences) for 15-18 h at 4°C, then incubated with fluorescent conjugated secondary antibody for 40-50 min at RT. DAPI was used to stain nuclei. Spheroid structures were imaged and analyzed using Olympus IX81 confocal microscopy systems [55 (link)].