Ab6946

As previously described (25 ), asynchronous DMS114 and H524 cells were sequentially labelled with 20 μM IdU for 20 min and 50 μM CldU for 20 min. To preserve long genomic DNA fibers, cells were embedded in low melting point agarose plugs and incubated in cell lysis buffer with proteinase K at 50°C for overnight. Washed plugs with TE buffer, and then melted plugs in 0.1M MES (pH 6.5) at 70°C for 20 min. Agarose was subsequently degraded by adding 2 μl of β-agarase (New England Biolabs). DNA fibers were then stretched onto salinized coverslips (Genomic Vision, cov-002-RUO) using an in-house combing machine. Combed DNA on coverslips was then baked at 60 °C for 2 hours and denatured in 0.5 N NaOH for 20 min. IdU, CldU and single-strand DNA were detected using a mouse antibody directed against BrdU (IgG1, Becton Dickinson, 347580, 1:25 dilution), a rat antibody directed against BrdU (Accurate chemical, OBT0030, 1:200 dilution) and a mouse antibody directed against single-stranded DNA (ssDNA) (IgG 2a, Millipore, MAB3034, 1:100), respectively. The secondary antibodies used were goat anti-mouse Cy3 (Abcam ab6946), goat anti-rat Cy5 (Abcam, ab6565), and goat anti-mouse BV480 (Jackson ImmunoResearch, 115–685-166) for ssDNA. Slides were scanned with a FiberVision Automated Scanner (Genomic Vision). Replication signals on single DNA fibers were analyzed using FiberStudio (Genomic Vision).