Mouse anti brdu idu antibody

DNA fiber labeling was performed as previously described (Lossaint et al, 2013 (link); Ribeyre et al, 2016 (link)). Cells were labeled with 25 μM IdU, washed with warm media, and exposed to 50 μM CldU. Cells were lysed, and DNA fibers were stretched onto glass slides. The DNA fibers were denatured with 2.5 M HCl for 1 h, washed with PBS, and blocked with 2% BSA in PBS/Tween for 60 min. IdU replication tracts were revealed with a mouse anti-BrdU/IdU antibody from BD Biosciences (347580) and CldU tracts with a rat anti-BrdU/CldU antibody from Eurobio (ABC117-7513). The following secondary antibodies were used: Alexa Fluor 488 anti-mouse antibody (A11001; Life) and Cy3 anti-rat antibody (712-166-153; Jackson ImmunoResearch). Replication tract lengths were analyzed using ImageJ software. For statistical analysis, we used a non-parametric Mann–Whitney test with Prism software.

DNA fiber labeling scheme to visualize replication fork degradation was performed as previously described^{65 (link)}. Briefly, cells were labeled with 25 µM IdU, washed with warm media, exposed to 50 µM CldU, washed again with warm media, and treated with 5 mM hydroxyurea for 4 h. Cells were lysed and DNA fibers were stretched onto glass slides and then dried and fixed in methanol/acetic acid (3:1) for 10 min. The DNA fibers were denatured with 2.5 M HCl for 1 h, washed with PBS, and blocked with 2% BSA in PBS-Tween for 60 min. IdU replication tracts were revealed with a mouse anti-BrdU/IdU antibody from BD Biosciences (347580; 1:100) and CldU tracts with a rat anti-BrdU/CldU antibody from Eurobio (ABC117–7513; 1:100). The following secondary antibodies were used: Alexa fluor 488 anti-mouse antibody (Life A21241; 1:100) and Cy3 anti-rat antibody (Jackson ImmunoResearch 712-166-153; 1:100). Fibers were visualized and imaged by Carl Zeiss Axio Imager Apotome using ×40 Plan Apo 1.4 NA oil immersion objective and acquired using Zeiss Zen 3.1 software. Replication tract lengths were analyzed using ImageJ software.