Coolcube1 camera

B cell labeling was performed with 50 µM CldU (Sigma) for 30 min, followed by 250 µM IdU (Sigma) for an additional 30 min. Cells were harvested in ice-cold 1 x PBS, lysed using spreading buffer (0.5% SDS, 200 mM Tris-HCl pH 7.4, 50 mM EDTA), and stretched along super-frost microscope slides, allowing DNA to spread. Slides were air-dried, fixed in methanol and acetic acid (3:1) for 5 min, rehydrated in PBS for 10 min, and denatured with 2.5 M HCl for 30 min at room temperature. Slides were rinsed in PBS and blocked in PBS/0.1%Triton-X100/ 5% BSA for 1 h at room temperature. Slides were then stained using primary rat anti-BrdU (1:100, BU1/75 (ICR1), Abcam, ab6326) and mouse anti-BrdU (1:100 B44, Beckton Dickinson, 347580) antibodies for 1.5 h and then with corresponding secondary goat anti-rat Alexa594 and anti-mouse Alexa488 (A-11007 and A-11001, Thermo Fisher Scientific) antibodies (1:300) for 45 min. Slides were then mounted in Prolong Gold Antifade reagent (Invitrogen) and left to dry overnight. DNA fibers were analyzed using a Carl Zeiss AxioImager Z2 equipped with a CoolCube 1 camera and a ×40/0.75 objective. All images were processed with the ISIS fluorescence imaging system, and DNA fibers were measured using ImageJ. Statistical analyses were carried out using GraphPad Prism and Mann–Whitney non-parametric test.