Fitc labeled antirabbit iggs

Nonmalignant human mammary epithelial cells (MCF10A, ATCC) were cultivated in minimum essential medium (MEM, Invitrogen), supplemented with bovine pituitatry hormone (13 mg/mL), hydrocortisone (0.5 mg/mL), hEGF (10 µg/mL), insulin (5 mg/mL), and cholera toxin (100 ng/mL) (Invitrogen). Cell culture was performed in Permanox plastic 8-well Lab-Tek chamber slides (Nalge Nunc International Corporation) at 37C, with 95% humidity and 5% CO₂. The cells were grown to a confluent layer prior to irradiation. After three washing steps with PBS, cells were blocked with 0.2% BSA for 15 min, incubated with the primary antibodies for 15 min, washed with PBS, and incubated with the secondary antibodies for 15 min. The primary antibodies were a rabbit polyclonal anti 53BP1 antibody (1:100 from 1 mg/mL, Bethyl Laboratories) and a mouse monoclonal to phosphohistone H2AX antibody (1:100 from 1 mg/mL, clone JBW301, Upstate Cell Signaling Solutions Inc.). The corresponding secondary antibodies were either FITC-labeled antirabbit IgGs, or T-Red-labeled antimouse IgGs (Molecular Probes Invitrogen). After a further washing step with PBS, the cells were counterstained with DAPI. All steps were performed at room temperature.