Rabbit anti topbp1

U2OS cells grown on cover slips were washed three times with PBS, fixed with 4% formaldehyde in PBS for 10 min, permeabilized with 0.5 % Triton X-100 for 10 min, incubated with signal enhancer (Image-iT FX; Invitrogen) for 30 min, and probed with primary antibodies: rabbit anti-Mcm3 (1:200) (gift from B. Stillman), rabbit anti-TopBP1 (1:100) (Bethyl), rat anti-BrdU (1:100) (Abcam) or rat anti-Cdc45 C45-3G10 (Bauerschmidt et al, 2007) (1: 50) for 2 h. In Figure 4, cells were fixed and permeabilized as described in the figure legend. For BrdU staining, samples were incubated with primary antibody and 125 U/ml benzonase (Novagen), washed with PBS, and incubated with secondary antibodies (Invitrogen) AlexaFluor 633 anti-rat and AlexaFluor 555 anti-rabbit diluted 1:100 for 1 h. All antibodies were diluted in 10% FBS-PBS. Nuclear DNA in the cells was counterstained with DAPI for 20 min. Cover slips were mounted in ProLong antifade reagent (Molecular Probes, Eugene, OR). Data were collected using an Olympus FV-1000 confocal microscope equipped with three lasers giving excitation lines at 633, 543, 488 nm and UV light at a resolution of 1,024 by 1,024 pixels utilizing a 63x oil immersion objective. The data from the channels were collected sequentially using the appropriate band-pass filters built into the instrument. Data sets were processed using the FV10-ASW 1.6 Viewer software.