Alexa fluor 568 goat anti mouse igg secondary antibody

For FANCD2 foci visualization, U2OS cells grown in chamber slides (BD Falcon) were pre-extracted with PBS/ 0.3 % Triton X-100 and fixed with 4 % paraformaldehyde in PBS. Following three washes with PBS, cells were blocked with 2 % bovine serum albumin (BSA, Sigma-Aldrich). Cells were incubated with an anti-FANCD2 primary antibody (1: 500, Novus Biologicals) in PBS/ 1 % BSA for 2 h at RT, washede three times in PBS, and incubated with 1:1000 Alexa Fluor^® 568 goat anti-mouse IgG secondary antibody (Molecular Probes) for 1 h at RT. Cells were mounted with DAPI-containing mounting medium (Vector Lab), and images were captured using a Zeiss LSM 510 inverted confocal microscope with Zeiss LSM software. Images were processed using Adobe Photoshop CS6.

For GFP fluorescence, cells were grown on coverslips, UVC irradiated, fixed with 4% paraformaldehyde for 10 min at RT, washed three times with PBS and mounted with DAPI-containing mounting medium (Vector Lab). For γH2AX foci, fixed cells were permeabilized with PBS/ 0.3% Triton X-100 and blocked with 2% bovine serum albumin (BSA, Sigma-Aldrich). Cells were incubated with an anti-γH2AX primary antibody (1: 500, Millipore) in PBS/ 1% BSA and 1:1000 Alexa Fluor^® 568 goat anti-mouse IgG secondary antibody (Molecular Probes). Images were captured using a Zeiss LSM 510 inverted confocal microscope with Zeiss LSM software.