Hcx pl apo 40 0.75 na objective

Cells grown onto round coverslips were treated as described in the figure and incubated with 25 μM EdU for the last 30 min. The coverslips were then washed twice with PBS and cells were pre-extracted with CSK buffer (10 mM PIPES pH 7, 0.1 M NaCl, 0.3 M sucrose, 3 mM MgCl₂) for 5 min 4 °C. After fixation with formaldehyde for 10 min at room temperature, a click reaction was performed on the coverslips (100 mM Tris pH 8, 10 mM CuSO₄, 2 mM Na-l-ascorbate, 50 μM biotin-azide-AF488) for 1.5 h at 37 °C. Then, the coverslips were washed with PBS and DNA was stained with DAPI (1 mg ml⁻¹ in PBS) for 10 min at room temperature, mounted with Prolong Gold Antifade (Thermo Fisher Scientific) and visualized under a fluorescence microscope (DM6000 B Leica microscope with a HCX PL APO 40 0.75 NA objective). EdU intensity was assessed in at least 250 EdU-positive nuclei per condition per experiment using CellProfiler (v.3.1.9).

Cells were pulse-labeled (20 min) with 50 μm CldU followed by 250 μm IdU (20 min) prior to cell harvesting and lysis in 0.2 m Tris (pH 7.4), 50 mm EDTA, and 0.5% SDS. Stretched DNA fibers were prepared as described previously (5 (link)). For immunodetection of labeled tracks, fibers were incubated with anti-CldU (rat monoclonal anti-BrdU, Abcam, AB6326), anti-IdU (mouse monoclonal anti-BrdU, BD Biosciences, 347580), and anti-single-stranded DNA (Millipore, MAB3034) for 1 h at room temperature in a humidity chamber. Alexa Fluor-conjugated secondary antibodies (Invitrogen/Molecular Probes, A-11007, A-21121, and A-21241) were used for 30 min at room temperature. Images were obtained with a DM6000 B Leica microscope with a HCX PL APO ×40 0.75 NA objective. The conversion factor used was 1 μm = 2.59 kb (3 (link)). Signals were measured and quantified using ImageJ software (41 (link)). For FR, 250–350 forks (red–green tracks) were measured. For IOD, 15–50 measurements between two adjacent origins on intact fibers were taken. For origin firing, origins labeled during the first pulse (green–red–green structures) were quantified as percentage of all structures containing red (>500 total structures scored in each case).