Click it alexa fluor 647 azide

For Cyclin F and V5 co-staining, cells grown on coverslips were washed with PBS, fixed with formaldehyde 4% for 10 min, permeabilized with PBS containing 0.3% Triton X-100 for 10 min and blocked for 1 h in PBS containing 0.1% Triton X-100, 3% BSA before incubation with primary antibodies Cyclin F (sc-952, SCBT, 1/500 dilution), V5 (NB100–62264, Novus, 1/500 dilution), Phospho-RPA32 S4/S8 (A300-245A, Bethyl Labs, 1/1,000 dilution). For proliferating-cell nuclear antigen (PCNA) and H3S10ph co-staining, cells grown on coverslips were washed with PBS, then extracted for 1 min in cold pre-extraction buffer (0.5% Triton X-100, 20 mM HEPES, pH 7.5, 300 mM sucrose, 50 mM NaCl and 3 mM MgCl₂), washed with PBS and fixed for 15 min in methanol at −20 °C. After additional washing in PBS cell were incubated with PCNA (Santa Cruz #SC-56, 1/1,000 dilution) and H3S10ph (Millipore, 06–570, 1/1,000 dilution) antibodies. Secondary antibodies were from donkey and conjugated with Alexa Fluor fluorochromes (Invitrogen, 1/1,000 dilution). EdU was detected using Click-IT Alexa Fluor 647 azide (Invitrogen) and Click-IT cell reaction buffer kit (Invitrogen #C10269). Images were acquired using an Axiovert 200M confocal microscope equipped with an LSM510 laser module (Zeiss).