Tritc conjugated anti rabbit antibody

Ten thousand OVTOKO and OVISE cells, which overexpressed FLAG-MCM2-FL and 3×FLAG-MCM2-ΔN, were cultured on Falcon^® 4 Well Culture Slides (BD Falcon NJ, USA). Cells were fixed in 100% ethanol at −20° C for 20 min and then incubated with rabbit monoclonal anti-FLAG antibody (Sigma) at a 1:100 dilution in PBS for 1 h at room temperature. Then, they were stained with a TRITC-conjugated anti-rabbit antibody (Dako Cytomation, Glostrup, Denmark) at a 1:100 dilution for 20 min at room temperature. Slides were washed three times with PBS and mounted with mounting medium (Dako Cytomation) containing 4′,6-diamidino-2-phenylindole (DAPI, Abbott Molecular Inc., Des Plaines, IL, USA). Images were acquired using a FV1200 laser-scanning microscope (OLYMPUS, Tokyo, Japan) with a 1,000× objective.

HepG2 and Hep3B cells were grown on glass coverslips, fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. Coverslips were incubated for 1 h in phosphate-buffered saline containing 0.1% bovine serum albumin and a mixture of antibodies. After two washes in phosphate-buffered saline, the cells were incubated for 1 h with TRITC-conjugated anti-rabbit antibody (1:100 dilutions) (DAKO, Glostrup, Denmark). DNA was labeled using the Hoechst 33258 dye (1 μg ml⁻¹). Cells were examined using laser scanning microscopy (LCM 510; Carl Zeiss, Jena, Germany) as previously described.^{21 (link)}