2.6. Immunocytochemistry (ICC)

Cell cultures for ICC were washed with PBS before fixation with ice-cold methanol for 10 min. Residual methanol was removed by washing with PBS and air-drying. Cells were rehydrated prior to staining procedures with PBS and blocked with 6% bovine serum albumin (BSA) and 2% normal goat serum (NGS) in PBS for 30 min. Primary anti-cytochrome C antibodies (D18C7, Cell Signaling, Danvers, MA, USA) and mouse anti β-actin antibodies (AC-74, Sigma-Aldrich, St. Louis, MO, USA) in 1% BSA at 1:600 dilution were incubated for 4 h at RT or overnight at 4 °C. Samples were washed three times for 10 min with PBS and finally incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-Rabbit IgG secondary antibodies (Life Technologies, Carlsbad, CA, USA) at 1:600 dilution. Samples stained without primary antibody were used as secondary controls.

For propidium iodide (PI)/Hoechst 3032 staining, cells plated at 4 × 10⁴ per well after 72 h of culture in experimental conditions were processed by addition of propidium iodide (P3566 Invitrogen, Waltham, MA, USA) at 20 µg/mL and Hoechst 33342 (H3570 Invitrogen) at 5 µg/mL. After the addition of dyes, the cells were incubated at room temperature in the dark for 10 min, and fluorescent microscopy images were captured in triplicate or more per treatment on an Olympus IX71 inverted microscope.