Cells were pulse-labelled with 25 μM 5-chloro-2′-deoxyuridine (CldU) and 250 μM 5-iodo-2′-deoxyuridine (IdU) at specified times, with or without treatment as reported in the experimental schemes. Cells were or not pre-treated with the transcription inhibitor 5,6-dichloro-1-ß-d -ribofurosylbenzimidazole (DRB 50 μM for 1 h). DNA fibres were prepared and spread out as previously reported (35 (link)). For immunodetection of labelled tracks the following primary antibodies were used: anti-CldU (rat-monoclonal anti-BrdU/CldU; BU1/75 ICR1 Abcam) and anti-IdU (mouse-monoclonal anti-BrdU/IdU; clone b44 Becton Dickinson). The secondary antibodies were: goat anti-mouse Alexa Fluor 488 or goat anti-rat Alexa Fluor 594 (Molecular Probes). The incubation with antibodies was accomplished in a humidified chamber for 1 h at RT.
Images were acquired randomly from fields with untangled fibres using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks were measured using the Image-Pro-Plus 6.0 software, and values were converted into kilobases using the conversion factor 1 μm = 2.59 kb as reported (35 (link)). A minimum of 100 individual fibres were analysed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using Graph Pad Prism Software.
Images were acquired randomly from fields with untangled fibres using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks were measured using the Image-Pro-Plus 6.0 software, and values were converted into kilobases using the conversion factor 1 μm = 2.59 kb as reported (35 (link)). A minimum of 100 individual fibres were analysed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using Graph Pad Prism Software.
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