4.5. Immunofluorescence Analysis

For nuclear detection of Ki-67, control and treated cells were fixed in acetone/methanol 1/1 (v/v) for 10 min at room temperature and air-dried. After 1 h of preincubation with PBS containing 10% of AB human serum, the cells were incubated for 1 h at room temperature with FITC-conjugated mouse anti-human Ki-67 (BD Biosciences). The nuclei were stained with Hoechst 33258 (Sigma-Aldrich) at 37 °C for 15 min. The samples were mounted on glass cover slips with glycerol/PBS (2:1) and observed by intensified video microscopy (IVM) with an Olympus Microphot fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a Zeiss CCD camera.