Cells were pulse-labelled with 25 μM CldU followed by 250 μM IdU for 20–40 min each. After labelling, cells were trypsinized and lysed in spreading buffer (200 mM Tris-Hcl pH 7.4, 50 mM EDTA and 0.5% SDS) before spreading on a microscope slide (Menzel-Gläser,Superfrost). Slides were fixed in methanol: acidic acid 3:1. Before immunodetection, slides were treated with 2.5 M HCl for 1 hr and 15 min. To detect CldU and IdU labelled tracts slide were incubated for 1 hr with rat-anti-Brdu (Clone BU1/75, Novus Biologicals; 1:500) and mouse-anti-BrdU (clone B44, Becton Diskinson; 1:750), respectively. Subsequently, slides were fixed with 4% paraformaldehyde for 10 min and incubated with Alexa 488-labeled goat-anti-mouse and Alexa 555-labeled goat-ant-rat (Molecular probes; 1:500) for 1 hr and 30 min. Pictures were taken with a Zeiss AxioObserver Z1 inverted microscope using a 63x lens equipped with a cooled Hamamatsu ORCA AG Black and White CCD camera and track lengths were analyzed with ImageJ software. Replication track lengths were calculated using the conversion factor 1 μm = 2.59 kb (Jackson and Pombo, 1998 (link)). The 1-way ANOVA (nonparametric Kruskal-Wallis test) was used for statistical analyses.
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