Anti rabbit igg alexafluor 555

The cells were washed and fixed as follows. For 53BP1 staining, the cells were treated with CSK buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl and 3 mM MgCl₂) for 1 min, then with 0.5% Triton X-100 in CSK buffer for 1 min, and finally with 4% PFA in PBS for 10 min at room temperature; for Cyclin A, the γH2AX and CldU staining cells were treated with 4% PFA for 10 min and 0.3% Triton Χ-100 in PBS for 5 min at room temperature; the S9.6 staining cells were treated with methanol for 10 min on ice and 0.5% Triton X-100 in PBS for 5 min at room temperature. The samples were blocked with 3% BSA/10% foetal bovine serum in PBS. Primary antibodies were mouse anti-phospho-Histone H2AX (Ser139) (JBW301, Millipore 05-636, 1:1000), rabbit anti-53BP1 (Bethyl A300-272A, 1:30,000), mouse anti-Cyclin A (6E6, Thermo Scientific MS1061, 1:50), rat anti-CldU (BU1/75, AbD Serotec OBT0030G, 1:250) and mouse anti-RNA/DNA hybrid (S9.6, gift from Professor Richard Gibbons, hybridoma supernatant 1:100). The secondary antibodies were anti-mouse IgG AlexaFluor 488 and anti-rabbit IgG AlexaFluor 555 MolecularProbes). DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI) and images acquired as above. For the quantification of nuclear S9.6 intensity, ImageJ was used to generate nuclear masks based on DAPI staining and mean S9.6 fluorescence intensities per pixel were quantified per nucleus.

Cells were washed and fixed as follows. γH2AX and 53BP1: CSK buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl and 3 mM MgCl₂) for 1 min, 0.5% Triton X-100 in CSK buffer for 1 min and 4% PFA for 10 min at room temperature; Cyclin A and CldU: 4% PFA for 10 min and 0.3% Triton Χ-100 in PBS for 5 min at room temperature; S9.6: methanol for 10 min on ice and 0.5% Triton X-100 in PBS for 5 min at room temperature. RNase H (New England Biolabs) was used at 0.05 U μl⁻¹ for 36 h at 37 °C. RNase A (Invitrogen) was used at 0.05 ng μl⁻¹–2 μg μl⁻¹ for 2 h at 37 °C. Samples were blocked with 3% BSA/10% fetal bovine serum. Primary antibodies were mouse anti-phospho-Histone H2AX (Ser139) (JBW301, Millipore 05-636, 1:1,000), rabbit anti-53BP1 (Bethyl A300-272A, 1:30,000), mouse anti-Cyclin A (6E6, Thermo Scientific MS1061, 1:50), rat anti-CldU (BU1/75, AbD Serotec OBT0030G, 1:250) and mouse anti-RNA/DNA hybrid (S9.6, gift from Professor Richard Gibbons, hybridoma supernatant 1:100). Secondary antibodies were anti-mouse IgG AlexaFluor 488 and anti-rabbit IgG AlexaFluor 555 (Molecular Probes). DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI) and images acquired as above. For quantification of nuclear S9.6 intensity, ImageJ was used to generate nuclear masks based on DAPI staining and mean S9.6 fluorescence intensities per pixel were quantified per nucleus.