Samples grown over Nunc Lab-Tek chambers (Thermo Fisher Scientific, Waltham, MA, USA) were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS and blocked with PBS 1% BSA-0.3% Triton for 60 min. Samples were then incubated overnight at 4 °C with primary antibody against BAX-6A7 (BD Bioscience, Franklin Lakes, NJ, USA), activated Caspase 3 (Cell Signaling, Danvers, MA, USA) or γH2AX (Cell Signaling, Danvers, MA, USA) in PBS 1% BSA-0.3% Triton.
After three washes with PBS, samples were incubated with the corresponding FITC-conjugated (Activated Caspase-3 or γH2AX) or TRITC-conjugated (BAX-6A7) secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Samples were washed three times with PBS and cell nuclei were finally stained with DAPI (2 μg/mL). Images were captured using a ZEISS Axio Observer (ZEISS, Oberkochen, Germany) microscope and analyzed using the Carl Zeiss Microscopy GmbH’s ZEN 3.0 software.
After three washes with PBS, samples were incubated with the corresponding FITC-conjugated (Activated Caspase-3 or γH2AX) or TRITC-conjugated (BAX-6A7) secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Samples were washed three times with PBS and cell nuclei were finally stained with DAPI (2 μg/mL). Images were captured using a ZEISS Axio Observer (ZEISS, Oberkochen, Germany) microscope and analyzed using the Carl Zeiss Microscopy GmbH’s ZEN 3.0 software.