Mouse anti brdu clone b44

Replication progression was analysed on DNA fibres as described previously (10 (link)). Briefly, approximately 15 × 10⁴ cells were labelled with 25 μM CldU for 20 min followed by 250 μM IdU for a further 20 min. For fork stalling assays, a pulse of 20 J/m² UV-C was given in between the two labels. Cells were lysed and DNA was spread down slides using gravity before being fixed with 3:1 methanol:acetic acid. After rehydration, fibres were stained with antibodies to the specific labels, rat anti-BrdU [BU1/75 (ICR1)] (Abcam ab6326), mouse anti-BrdU Clone B44 (BD 347580), anti-rat Alexa Fluor 488 (A21208), anti-mouse Alexa Fluor 594 (A31624) (Invitrogen Molecular Probes). Slides were mounted with Fluoromount (Sigma-Aldrich) and imaged and quantified as described above.

DNA fiber analysis was performed essentially as in ref. 47 . Briefly, ongoing DNA synthesis was labeled with the indicated nucleotide analogs, cells were washed with PBS, lysed with lysis buffer (0.5% SDS, 200 mM Tris–HCL (pH 7.4) and 50 mM EDTA) and spread on glass slides by tilting. After drying, the slides were fixed in methanol: acetic acid (3:1), dried, and rehydrated in PBS. DNA was denatured by incubating the slide in 2.5 N hydrochloric acid for 1 h. After neutralization in PBS, samples were blocked in blocking buffer (10% NGS in PBS), and stained with primary antibodies (Mouse Anti-BrdU Clone B44 (BD, #347580) for IdU, Abcam ab6326 for CldU, both 1:50 in the blocking buffer), washed with PBS-1%Tween20, incubated with fluorescently labeled secondary antibodies (Goat anti-mouse AlexaFluor 488 (Invitrogen, #A-11001), Goat anti-rat AlexaFluor 594 (Invitrogen, #A-11007), both 1:150 in blocking buffer). After extensive washes with PBS-Tween20 and PBS, slides were mounted with Prolong Diamond Antifade mounting medium (Invitrogen, #P36961). Samples were imaged using Olympus BX61 fluorescence microscope at 60x magnification; image analysis was performed using Fiji (ImageJ) software. At least 100 tracks per sample were analyzed.

We have used pulse-labeling conditions that support DNA polymerization at normal rates according to [31 (link)]. Labelled-cells were harvested, lysed and DNA spreading, and immunostaining were performed as previously described [58 (link)]. For CIdU detection, a rat anti- Bromodeoxyuridine (BrdU, clone BU1/75(ICR1); AbD Serotec) were used in a dilution 1:500 and detected with an Alexa-555 conjugated goat anti-rat IgG (A-21434; Molecular Probes, Eugene, Oregon, USA). For IdU detection, a mouse anti-BrdU [(clone B44); BD, Franklin Lakes, NJ, USA] were used in a dilution 1:500 and detected with an Alexa-488 conjugated F(ab')₂ fragment of goat anti-mouse IgG (A-11017; Molecular Probes). Antibody dilutions and washes were performed in PBS containing 1% BSA and 0.1%Tween 20. Coverslips were mounted in Fluoroshield medium (F6182; Sigma-Aldrich).