Anti rat igg alexafluor 555

Cells were pulse labeled with 25 μM CldU and 250 μM IdU for 15 or 20 min each and harvested. DNA fiber spreads were prepared by spotting 2 μL of cells (5^∗10⁵ cells per ml in PBS) onto microscope slides followed by lysis with 7 μL of 0.5% SDS, 200 mM Tris-HCl pH 7.4 and 50 mM EDTA. Slides were tilted and DNA spreads fixed in methanol/acetic acid (3:1). HCl-treated fiber spreads were incubated with rat anti-bromodeoxyuridine (detects CldU, abcam, ab6326, 1:1,200) and mouse anti-bromodeoxyuridine (detects IdU, B44, Becton Dickinson, 1:500) for 1 h and incubated with anti-rat IgG AlexaFluor 555 and anti-mouse IgG AlexaFluor 488 (both at 1:500, Molecular Probes) for 1.5 h. Images were acquired using a Zeiss AxioImager M1, equipped with a Hamamatsu digital camera and the Volocity software (Perkin Elmer). Fiber length was analyzed using ImageJ (https://imagej.nih.gov/ij/). For fork speed analysis, during each independent experiment, a minimum of 300 fibers were measured per condition. Fork asymmetry was measured as a percentage of the length ratio of the shortest to the longest fiber of first label origin fibers.

After appropriate treatment with control or CNOT1 siRNA for 72 h, the cells were pulse-labelled with 25 μM CldU and 250 μM IdU for 20 min and harvested. DNA fibre spreads were prepared by spotting 10³ cells onto microscope slides followed by lysis with 7 μL 0.5% SDS, 200 mM Tris-HCl pH 7.4 containing 50 mM EDTA. The DNA spreads were fixed in methanol–acetic acid (3:1) and then treated with HCl. After washing with PBS, HCl-treated fibre spreads were incubated with rat anti-bromodeoxyuridine (detects CldU, AbD Serotec) and mouse anti-bromodeoxyuridine (detects IdU, Becton Dickinson, East Rutherford, NJ, USA) antibodies for 1 h, fixed with 4% paraformaldehyde (PFA) in PBS to increase staining intensity and incubated with anti-rat IgG AlexaFluor 555 and anti-mouse IgG AlexaFluor 488 antibodies (Molecular Probes) for 1.5 h. The images were acquired using a Nikon E600 microscope (Nikon, Tokyo, Japan) with a Nikon Plan Apo × 60 (1.3 numerical aperture) oil lens, a Hamamatsu digital camera (C4742-95) and the Volocity acquisition software (version v4.1) (Perkin Elmer, Milan, Italy). The images were analysed using ImageJ; in each independent experiment, at least 300 fibres were measured per condition.