Analysis of replicating DNA were performed according to the methods described previously32 (link). In brief, cells were pulse-labeled with 50 μM CldU for 15 min, followed by second labeling with 50 μM IdU for 15 min before analysis. Cells were harvested and DNA fibers were stretched onto glass slides in a DNA lysis buffer (200 mM Tris-HCl at pH 7.4, 0.5% SDS, 50 mM EDTA). Single replicating DNA was detected with rat anti-CldU (Novus), mouse anti-IdU (Becton Dickinson) and anti-single-strand DNA (EMD Millipore) antibodies. Images were captured using the Nikon Eclipse TiE microscope and processed using NIS Elements AR imaging software.
Mouse anti idu
Manufactured by BD
Mouse anti-IdU is a primary antibody that binds to the incorporated DNA analog 5-iodo-2'-deoxyuridine (IdU). It is used in immunodetection techniques to label and visualize IdU-incorporating cells.
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Lab products found in correlation
3 protocols using mouse anti idu
1
Replicating DNA Analysis Protocol
2
Replicating DNA Analysis Protocol
3
Measuring DNA Replication Rates
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DNA replication rates were measured by labeling DNA fibers based on a protocol described earlier (38 (link)). Cells at 50–70% confluency were labeled with 20 μM CldU (Sigma) for 30 min, followed by 100 μM IdU (Sigma) for 30 min. DNA fibers from lysed cells were allowed to stretch and airdry for 40 min at room temperature. After fixing, DNA was denatured and blocked with 10% goat-serum/PBS-T. Slides were incubated with rat anti-CldU (Abcam) and mouse anti-IdU (Becton Dickinson), washed, incubated with goat anti-rat IgG Alexa Fluor 594 (Invitrogen) and goat anti-mouse Alexa Fluor 488 Plus (Invitrogen) and imaged on a Keyence BZ-X fluorescence microscope with a CFI Achromat 60×/0.8 objective. Fibers showing a red label extended by a green label were analyzed for each cell line using ImageJ and the length of the green label was measured. DNA fiber analysis was performed at least twice for every cell line and a representative analysis of a minimum of 100 fibers is shown.
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