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.
Protocol detail
DNA Fiber Analysis of CldU and IdU Incorporation
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BU1/75 ICR1
goat anti-mouse Alexa Fluor 488
goat anti-rat Alexa Fluor594
Eclipse 80i Nikon Fluorescence Microscope
A fibers
Alexa fluor 488
Antibodies
Brdu
Cells
Clone
Deoxyuridine
Fluorescence microscope
Goat
Iodo
Mouse
Prism
Pulse
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Variable analysis
independent variables
- Treatment (with or without as reported in the experimental schemes)
- Length of labelled tracks
- Pulse-labelling with 50 μM 5-chloro-2′-deoxyuridine (CldU) and 250 μM 5-iodo-2′-deoxyuridine (IdU)
- Antibodies used for immunodetection (anti-CldU and anti-IdU)
- Incubation with antibodies in a humidified chamber for 1 h at 37°C
- Imaging using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system
- Measurement of labelled track length using Image-Pro-Plus 6.0 software
- Minimum of 100 individual fibers analyzed for each experiment
- Statistics calculated using GraphPad Prism Software
- No positive or negative controls were explicitly mentioned in the provided information.
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