Rat anti brdu monoclonal antibody

DNA fiber assay was performed as described in(Bellelli et al., 2018 (link)). Briefly, eHAP ALC1^+/+ and ALC1^−/− transfected with control siRNA or siRNA targeting BRCA1 or BRCA2, were pulse labeled with 20 μM CldU for 20 min and subsequently with 200 μM IdU for 20 min. After tripsinization and counting, cells were resuspended at a concentration of 5x 10⁵ in PBS and 2.5 μL of cell suspension were spotted on glass slides and lysed with 7.5 μL of a buffer containing 0.5% SDS, 200 mM Tris-HCL, pH 7.4, and 50 mM EDTA. Slides were then tilted to allow a stream of DNA to move slowly toward the botton of the slide, briefly air-dried and then fixed in methanol/acetic acid (3:1) (15 min at R.T). Slides were subsequently denatured in HCl 2,5 M (30 min R.T.), extensively washed in dH2O and PBS, blocked in 1% BSA/PBS (30 min R.T.) and incubated with rat anti-BrdU monoclonal antibody (1:1000 overnight; AbD Serotec) and subsequently with mouse anti-BrdU monoclonal antibody (1:500 1 h R.T.; Becton Dickinson). After incubation with a mixture of Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 45 min R.T.; Invitrogen) slides were mounted in PBS/Glycerol 1:1 and finally examined using Axio Imager.M2 (ZEISS) with 63x oil immersion objective and the Volocity 6.3 software.

MCF7 cells were transfected with 100 nM hSSB1 siRNA or control siRNA (Allstars negative control siRNA, Qiagen) using Lipofectamine 2000 according to manufacturer's instructions, and re-transfected with hSSB1 siRNA 24 h later. Twenty-four hours after the second transfection, cells were pulse labelled with 25 μM CldU for 20 min, washed three times with medium, incubated in 2 mM HU for 2 h, washed three times with medium and pulse labelled with 250 μM IdU for 1 h. Labelled cells were harvested and DNA fibre spreads prepared as previously described (23 (link)). CldU was detected by incubating acid treated fibre spreads with rat anti-BrdU monoclonal antibody (1:1000, AbD Serotec) for 1 h. Slides were fixed with 4% PFA (paraformaldehyde) and incubated with Alexa-Fluor 555-conjugated goat anti-rat IgG (1:500, Molecular Probes) for 1.5 h. IdU was detected using mouse anti-BrdU monoclonal antibody (1:1000, Becton Dickinson) over night at 4°C and Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:500, Molecular Probes) for 1.5 h. Fibres were examined using a Biorad Radiance confocal microscope with a 60× oil immersion objective. For quantification of replication structures, at least 250 structures per experiment were counted using the ImageJ software (National Institutes of Health, http://rsbweb.nih.gov/ij/). Statistical analysis was performed using a one-tailed paired t-test.

A3 Cells were exposed to either 0.1% DMSO or 10 μM TH5487 for the indicated times, pulse-labeled with 25 μM 5-chloro-2′-deoxyuridine (CldU) for 30 min, washed with medium and pulse-labeled with 250 μM 5-iodo-2′-deoxyuridine (IdU) for 30 min. Alternatively, control or OGG1 shRNA expression was induced with 1 μg/ml doxycyline for 48 h or 96 h before pulse labelling. CldU was detected by incubating acid-treated fiber spreads in Ibidi μ-Slide VI 0.4 (Ibidi, 80606) with rat anti-BrdU monoclonal antibody (AbD Serotec; cat# MCA2060), whereas IdU was detected using mouse anti-BrdU monoclonal antibody (BD Biosciences; cat# 347580) for 1 h at 37°C. Slides were fixed with 4% paraformaldehyde and incubated with goat anti-rat Alexa Fluor 555 or goat anti-mouse Alexa Fluor 488 for 1.5–2 hours. Fibers were examined using a Zeiss (Jena, Germany) LSM780 confocal laser scanning microscope with a 63× oil immersion objective. The lengths of red (AF 555) or green (AF 488) labeled patches were measured using the ImageJ software (National Institutes of Health; http://rsbweb.nih.gov/ij/) and arbitrary length values were converted into micrometers.

DNA fiber assay was essentially performed as described in Bellelli et al., 2018a (link)). Briefly, MEFs of the indicated genotypes were pulse labelled with 20 μM CldU for 20 min and subsequently pulse labelled with 200 μM IdU for 20 min. Cells were trypsinized, washed in PBS, counted and resuspended at a concentration of 5 × 10⁵ in PBS. 2.5 μL of cell suspension were spotted on clean glass slides and lysed with 7.5 μL of 0.5% SDS in 200 mM Tris-HCL, pH 7.4, 50 mM EDTA (10 min, R.T.). Slides were tilted (15° to horizontal), allowing a stream of DNA to run slowly down the slide, air dried and then fixed in methanol/acetic acid (3:1) for 15 min at R.T. Acid-treated slides (45 min R.T.) were blocked in 1% BSA/PBS for 45 min at R.T. and incubated with rat anti-BrdU monoclonal antibody (1:1,000 over night; AbD Serotec) and mouse anti-BrdU monoclonal antibody (1:500 1h R.T.; Becton Dickinson). After 3 washes in PBS, slides were incubated with a mixture of Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 R.T.; Invitrogen) for 45 min at room temperature and mounted in PBS/Glycerol 1:1. Fibers were then examined using Axio Imager.M2 (ZEISS) with 60x oil immersion objective and the Volocity 6.3 software. For quantification at least 500 replication structures were counted per experiment.