Alexa fluor 488 linked secondary antibody

Prior to immunofluorescence analysis, cells were fixed for 30 min with 4 % paraformaldehyde and permeabilized with 0.2 % Triton-X-100 for 10 min. The coverslips were incubated with primary antibodies at 1:100 dilutions overnight at 4 °C in a humidified chamber. Following incubation phosphate-buffered solution (PBS) washing steps, the appropriate Alexa Fluor 488-linked secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was applied for 2 h at room temperature. The cells were then rinsed with PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and images were taken on a fluorescence microscope.

Both γH2AX and 8-hydroxy-2-deoxyguanosine (8-OHdG) were chosen as cellular DNA damage markers. Following fixation, the γH2AX [39 (link)] and 8-OHdG [40 (link)] expressions were detected by antibody-based methods. For γH2AX measurement, Santa Cruz Biotechnology γH2AX antibody (Santa Cruz, CA, USA) (4 °C, 1 h) and Alexa Fluor^®488-linked secondary antibody (Cell Signaling Technology) were sequentially applied to fixed cells, and counterstained with 7AAD (5 μg/mL, 30 min). For 8-OHdG measurement, the 8-OHdG-FITC antibody (Santa Cruz Biotechnology) (4 °C, 1 h) was applied. In addition, their intensities were monitored by Accuri C6 flow cytometry.