Goat anti rat alexa 488 conjugated secondary antibody

Replicative cells were marked by pulse labeling for 30 min with 50 μM of CldU and then arrested for 6 h by treating with 2 mM ddC or 2 mM HU. After drug removal, cells were incubated with 50 μM of IdU in fresh complete medium for 60 min so that replication restart after stalling could be visualized. All the following steps were carried out at room temperature. Cells were fixed in 4% paraformaldehyde for 10 min and then permeabilized in 0.5% Triton X-100 (for TK6 cells) or 0.1% NP-40 (for DT40 cells) for 20 min. Cells were incubated with 2 M HCl for 45 min to denature the DNA and then blocked for 1 h with 5% FCS in PBS. Cells were immunostained for 1 h with the first primary antibody of rat monoclonal anti-BrdU [BU1/75, Abcam], washed with 0.05% PBST20 and then immunolabeled for 40 min with goat anti-rat Alexa 488-conjugated secondary antibody (Molecular Probes) to label CldU. Then, cells were immunostained for 1 h with the second primary antibody of mouse monoclonal anti-BrdU (Becton Dickinson). Cells were subsequently labeled for 40 min with goat anti-mouse Alexa 594 conjugated secondary antibody (Molecular Probes) to label IdU. Replication restart is represented by the overlap of CldU and IdU. Nuclei were counterstained with DAPI as in immunofluorescence microscopy assays.

Hela cells were grown on slides and pulse-labeled for 30 min with 50 μM of CldU. Cells were washed with PBS and then treated with 5 μM of Cpt for 2 hrs, washed with PBS and incubated with fresh medium. Cells were then grown in media containing 50 μM of IdU for 60 mins. Cells were then washed and fixed in 4% paraformaldehyde for 15 min and then permeabilized in 0.5% Triton X-100 for 15 min. Cells were incubated with 2 M HCl for 45 min to denature the DNA and then blocked for 30 min with 5% milk in PBS-T. To visualize CldU, cells were immunostained for 1 h with rat primary monoclonal antibody against BrdU [BU1/75, Abcam], washed with 0.05% PBS-T 20 and three times for 5 min then immunolabeled for 30 min with goat anti-rat Alexa 488-conjugated secondary antibody (Molecular Probes). To visualize IdU cells were immunostained for 40 mins at 4 °C with mouse anti-BrdU monoclonal antibody (Becton Dickinson). Cells were subsequently labeled for 30 min with goat anti-mouse Alexa 594 conjugated secondary antibody (Molecular Probes) to label IdU. Replication restart is represented by the co-localization of CldU and IdU. Nuclei were counterstained with DAPI for fluorescence imaging.