Alexa fluor 488 goat anti rabbit or rabbit anti mouse antibodies

For indirect immunofluorescence staining, cells were seeded on coverslips and fixed in 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100 (5 min on ice), coverslips were blocked in 1% BSA/PBS and incubated with the following primary antibodies diluited in 0.5% BSA/PBS: anti-H2AX phosphorylated on Ser139 (γH2AX) (Millipore), −53BP1, (Novus Biologicals), for 1h at room temperature. Coverslips were then washed 3 times in PBS and incubated with Alexa Fluor 488 goat anti-rabbit or rabbit anti-mouse antibodies (Invitrogen) for 45 min at room temperature. After DAPI counterstaining, coverslips were mounted in Glycerol/PBS (1:1) and observed with Axio Imager.M2 (ZEISS) using the Volocity 6.3 software. For EdU immunofluorescence analysis MEFs (passage 3) were labeled and processed using the Click-iT® EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher). Cells were pulse labeled for 30 min with 10 mM EdU and fixed in 4% paraformaldehyde, before being permeabilized in PBS-Triton 0.5% and washed in 1% BSA. Cells were then resuspended in Click-iT reaction cocktail containing Alexa Fluor® 488 Azide and incubated for 30 min at R.T. After being washed, cells were finally counterstained for DNA content by DAPI (1 mg/ml) and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).

For indirect immunofluorescence stainings, cells were seaded on coverslips and fixed in 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100 (5 min on ice), coverslips were blocked in 1% BSA/PBS and incubated with the following primary antibodies diluited in 0.5% BSA/PBS: anti-H2AX phosphorylated on Ser139 (gH2AX) (Millipore), −53BP1, (Novus Biologicals), for 1h at room temperature. Coverslips were then washed 3 times in PBS and incubated with Alexa Fluor 488 goat anti-rabbit or rabbit anti-mouse antibodies (Invitrogen) for 40 min at room temperature. After DAPI counterstaining, coverslips were mounted in Glycerol/PBS (1:1) and observed with Axio Imager.M2 (ZEISS) using the Volocity 6.3 software.