Alexafluor 594 goat anti rat igg h l

DNA replication rates were measured by labeling DNA fibers based on a protocol described earlier^{93 (link),94 (link)}. Briefly, cells at ~ 50 to 70% confluency were sequentially labeled with 20 μM CldU (Sigma) for 30 min and 100 μM IdU (Sigma) for 30 min. Cells were then resuspended in PBS at a concentration of 1 × 10⁶ cells/ml. Labeled cells diluted with unlabeled cells were allowed to air-dry on a microscopy slide and incubated with 10 μl of lysis buffer (0.5% SDS, 200 mM Tris–HCl, pH 7.4, 50 mM EDTA). Slides were inclined at 15° to stretch the fibers. DNA spreads were fixed by incubation with 3:1 methanol:acetic acid followed by denaturation with 2.5 N HCl for 70 min. Following this, the slides were blocked with 10% goat-serum/PBS-T (PBS+ 0.1% Triton X-100) for 1 h. This was followed by incubation with rat anti-BrdU (anti-CldU; Abcam) and mouse anti-BrdU(anti-IdU; Becton Dickinson) at a dilution of 1:100 and 1:50 respectively in 10% goat-serum/PBS-T(PBS+ 0.1% Triton X-100) for 2 h. Slides were washed with PBS and incubated with AlexaFluor 594 goat anti-rat IgG (H+L) (Invitrogen) and goat anti-mouse (H+L) AlexaFluor 488 Plus (Invitrogen) in the dark for 1 h. Images were acquired using a Keyence BZ-X Fluorescence microscope with a CFI Achromat 60×/0.8 objective.