Eclipse te confocal microscope

Cells were seeded into fluorodish (World Precision Instruments) and incubated overnight for adherence. After drug treatment, cells were fixed with 4% formaldehyde for 10 min at room temperature. Cells were then permeabilized using 0.2% Triton X-100 in PBS for 3 min. Cells were washed and blocked using 10% goat serum in PBS for 40 min. After three washes with PBS, cells were incubated overnight at 4 °C with primary antibodies (BRCA1, RAD51, and γH2AX) in PBS, followed by incubating with fluorescent secondary antibodies (Molecular Probes, Eugene, OR) for 2 h at room temperature. Cells were mounted with Vectashield containing DAPI and analyzed for focus formation [38 (link)], using a Nikon Eclipse TE confocal microscope.

First, the cells were seeded into FluoroDishes (World Precision Instruments) and incubated overnight for adherence. After treatment, cells were fixed with 4% formaldehyde for 10 min at room temperature. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 3 min. The cells were washed and blocked with 10% goat serum in PBS for 40 min. After three PBS washes, cells were incubated overnight at 4 °C with primary antibodies (RAD51: Cat No: 8349, Santa Cruz or pH2AX: Cat No: 05636, Millipore) in PBS, then incubated with a fluorescent secondary antibody (Molecular Probes, Eugene, OR) for 2 h at room temperature. The cells were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole and analyzed for foci formation using a Nikon Eclipse TE confocal microscope. A total of three independent experiments were performed.