PMC:2246177 / 11664-12257 JSONTXT

DNA synthesis assay Following 48 hours of transfection with siRNA, SKBr3 cells or BT-20 cells were serum starved for an additional 18 hours and were then incubated with 100 μM bromodeoxyuridine (BrdU) for 6 hours. Cells were fixed and permeabilized as previously described [12]. Cells were blocked in 20% goat serum/PBS for 20 minutes at 37°C, and then were incubated with anti-BrdU-Alexa-Fluor 594 (Molecular Probes, Carlsbad, CA, USA) for 1 hour at 37°C. BrdU incorporation was visualized using a Leica DM RBE Fluorescence microscope (model RS232C; Leica Microsystems, Bannockburn, IL, USA).