Specie specific fluorophore conjugated antibodies

Cells were grown in 35-mm coverslips and harvested at the indicated times after treatments. For 53BP1 IF, after further washing with PBS, cells were fixed with 4% PFA at RT for 10 min. Cells were subsequently permeabilized with 0.4% Triton-X100. Staining with mouse polyclonal anti-53BP1 (1:300, Millipore), γ-H2AX (1:1000 Santa Cruz Biotechnology) or rabbit polyclonal anti-Cyclin A (1:100 Santacruz), pS10H3 (1:1000 Santa Cruz Biotechnology) diluted in a 1%BSA/0,1% saponin in PBS solution, was carried out for 1 h at RT. After extensive washing with PBS, specie-specific fluorophore-conjugated antibodies (Invitrogen) were applied for 1 h at RT followed by counterstaining with 0.5 mg/ml DAPI. Secondary antibodies were used at 1:200 dilution. Images were acquired as greyscale files using Metaview software (MDS Analytical Technologies) and processed using Adobe Photoshop CS3 (Adobe). For each time point, at least 200 nuclei were examined and foci were scored at 40×.

Cells were grown in 35-mm coverslips and harvested at the indicated times after treatment. For 53BP1 IF, after further washing with PBS, cells were fixed with 4% PFA at RT for 10 min. Cells were subsequently permeabilized with 0.4% Triton-X100. Staining with mouse polyclonal anti-53BP1 (Calbiochem), rabbit monoclonal anti-γ-H2AX (Ser 139; Millipore, Billerica, MA) in a 1%BSA/0,1% saponin in PBS solution, was carried out for 1 h at RT. After extensive washing with PBS, specie-specific fluorophore-conjugated antibodies (Invitrogen) were applied for 1 h at RT followed by counterstaining with 0.5 mg/ml DAPI. Secondary antibodies were used at 1:200 dilution. Coverslips were analysed using a Leica DRMB fluorescence microscope equipped with a charge coupled device camera. The images were processed using the IAS 2000 Delta System software (Adobe, San José, CA).