Eclipse 80i fluorescence microscope

Cells were pulse-labeled with 25 μM chlorodeoxyuridine (CldU) and then labeled with 250 μM iododeoxyuridine (IdU) at the times specified, with or without treatment as reported in the experimental schemes. DNA fibers were prepared and spread out as previously described (12 (link)). For immunodetection of labeled tracks, the following primary antibodies were used: rat anti-CldU/BrdU (Abcam) and mouse anti-IdU/BrdU (Becton Dickinson). Images were acquired randomly from fields with untangled fibers using Eclipse 80i Nikon Fluorescence Microscope, equipped with a VideoConfocal (ViCo) system. The length of labeled tracks were measured using the Image-Pro-Plus 6.0 software, and values were converted into kilobase using the conversion factor 1 μm = 2.59 kb as reported (19 (link)). A minimum of 100 individual fibers were analyzed for each experiment and the mean of at least three independent experiments presented.

In situ PLA (DuoLink, Merck) was performed according to the manufacturer's instructions. To detect parental ssDNA-RAD52 interaction, cells were labelled with 50 μM IdU for 20 h, released in fresh DMEM for 2 h and then treated as indicated. After treatment, cells were permeabilized with 0.5% Triton X-100 for 10 min at 4°C, fixed with 3% formaldehyde/2% sucrose solution for 10 min, and then blocked in 3% BSA/PBS for 15 min. After washing with PBS, cells were incubated with the two relevant primary antibodies. The primary antibodies used were: rabbit polyclonal anti-RAD52 (Aviva 1:150), and an anti-IdU (mouse monoclonal anti-BrdU/IdU; clone b44 Becton Dickinson, 1:10). In control experiments, parallel samples were probed with each primary antibody alone. Samples were incubated with secondary antibodies conjugated with PLA probes MINUS and PLUS (DuoLink, Merck). Incubation with primary and secondary antibodies was accomplished in a humidified chamber for 1 h at 37°C. PLA probes MINUS and PLUS were ligated using two connecting oligonucleotides to produce a template for rolling-cycle and hybridisation with TRITC-labelled oligonucleotide. Samples were mounted in Prolong Gold anti-fade reagent with DAPI to counterstain nuclei. Images were acquired randomly using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Virtual Confocal (ViCo) system.

Cells were pulse-labelled with 50 μM 5-chloro-2′-deoxyuridine (CldU) and 250 μM 5-iodo-2′-deoxyuridine (IdU) as indicated, with or without treatment as reported in the experimental schemes. DNA fibers were spread out as previously reported (26 (link)). For immunodetection of labelled tracks the following primary antibodies were used: anti-CldU (1:60; rat-monoclonal anti-BrdU/CldU; BU1/75 ICR1 Abcam) and anti-IdU (1:10; mouse-monoclonal anti-BrdU/IdU; clone b44 Becton Dickinson). The secondary antibodies were goat anti-mouse Alexa Fluor 488 or goat anti-rat Alexa Fluor594 (Invitrogen). The incubation with antibodies was accomplished in a humidified chamber for 1 h at 37°C. Images were acquired randomly from fields with untangled fibers using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks was measured using the Image-Pro-Plus 6.0 software. A minimum of 100 individual fibers were analyzed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using GraphPad Prism Software.

Immunofluorescence microscopy was performed on cells grown on cover- slips as described previously (28 (link)). Nocodazole-treated cells were blocked and fix with PTEMF buffer (30 (link)). After blocking, coverslips were incubated for 1hat RT with the indicated antibodies. For detection of anti-BrdU, after permeabilization with 0,4%Triton-X 100/PBS, cells were denatured in HCl 2.5N for 45 min at RT. Alexa Fluor^® 488 conjugated-goat anti mouse and Alexa Fluor^® 594 conjugated-goat anti-rabbit secondary antibodies (Life Technologies) were used at 1:200. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI 1:4000, Serva). Coverslips were observed at 40× objective with the Eclipse 80i Nikon Fluorescence Microscope, equipped with a VideoConfocal (ViCo) system. Images were processed by using Photoshop (Adobe) program to adjust contrast and brightness. For each time point at least 200 nuclei were examined. Parallel samples incubated with either the appropriate normal serum or only with the secondary antibody confirmed that the observed fluorescence pattern was not attributable to artefacts.
Experiments for labeling cellular DNA with EdU (5-ethynyl-2′-deoxyuridine). EdU was added to the culture media (10 μM), for 30 min. Detection of EdU was performed used Click-iT EdU imaging Kits (Invitrogen).