Rat anti brdu bu1 75 icr1

Replication progression was analysed on DNA fibres as described previously (10 (link)). Briefly, approximately 15 × 10⁴ cells were labelled with 25 μM CldU for 20 min followed by 250 μM IdU for a further 20 min. For fork stalling assays, a pulse of 20 J/m² UV-C was given in between the two labels. Cells were lysed and DNA was spread down slides using gravity before being fixed with 3:1 methanol:acetic acid. After rehydration, fibres were stained with antibodies to the specific labels, rat anti-BrdU [BU1/75 (ICR1)] (Abcam ab6326), mouse anti-BrdU Clone B44 (BD 347580), anti-rat Alexa Fluor 488 (A21208), anti-mouse Alexa Fluor 594 (A31624) (Invitrogen Molecular Probes). Slides were mounted with Fluoromount (Sigma-Aldrich) and imaged and quantified as described above.

Cells were first pulse labeled for 20 min with 250 μm IdU (Sigma), washed twice with PBS, and then pulse labeled with 30 μm CldU (Sigma) for 20 min. Cells were then collected and scraped into ice-cold PBS and genomic DNA was extracted with CombHeliX DNA Extraction kit (Genomic vision) in accordance with the manufacturer’s instructions. DNA fibers were stretched on vinyl silane-treated glass coverslips (CombiCoverslips) (Genomic vision) with automated Molecular Combing System (Genomic Vision). After Combing, the stretched DNA fibers were dehydrated in 37 °C for 2 h, fixed with MeOH:Acetic acid (3:1) for 10 min, denatured with 2.5 m HCl for 1 h, and blocked with 5% BSA in PBST for 30 min. IdU and CldU were then detected with the following primary antibodies in blocking solution for 1 h at room temperature: mouse anti-BrdU (B44) (1:40) (BD) for IdU and rat anti-BrdU [Bu1/75 (ICR1)] (1:50) (abcam) for CldU. After PBS wash, fibers were than incubated with secondary antibodies anti-Rat-Alexa488 (1:50) (Invitrogen) and anti-mouse Alexa 568 (1:50) (Life Technologies) for 1 h at room temperature. DNA fibers were analyzed on LeicaDM18 microscope at ×100 and ImageJ was used to measure fiber length. For the experiment with transcription inhibitor, cells were first treated with DMSO or 50 μm cordycepin for 2 h before IdU labeling.

To visualize BrdU or PCNA, antigen retrieval was first performed using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) as previously described [38 (link),40 (link),45 (link)]. Sections were then incubated in the primary antibody (Rat anti-BrdU [BU1/75(ICR1)], Abcam, ab6326, Cambridge, United Kingdom and Accurate Chemical and Scientific, OBT0030, Westbury, NY; Mouse anti-PCNA (clone PC 10), P8825, Sigma Aldrich; Mouse anti-HuC/D (16A11), A21271, Invitrogen, Carlsbad, CA) overnight at 4°C. After washing with PBST, sections were incubated in secondary antibodies (Alexa Fluor-conjugated 488, 594, and 647 goat anti-primary, Thermo Fisher Scientific) for 1 hour at room temperature. Nuclei were stained with either DAPI (Thermo Fisher Scientific) or TO-PRO-3 (Thermo Fisher Scientific). Fluorescence images were captured using a Leica TCS SP5 or SP8 confocal microscope (Leica Microsystems, Werzler, Germany). To visualize EdU-labelled cells, a Click-iT Assay kit (Thermo Fisher Scientific) was used.