The immunocytochemistry assay evaluated the Bcl-2 expression (apoptosis resistance-related) and LC3 (autophagy marker). Thus, PC3 cells were treated in 6-well plates containing a coverslip at the bottom for cell adhesion. Cells were fixed in 4% methanol for 20 min, washed in TBS-T buffer, and blocked for nonspecific interactions with 3% albumin for 1 h. Then, primary antibodies Rabbit anti-Bcl-2 (Cell Signaling #3498, Massachusetts, USA) and Mouse Anti-LC3 B (Cell Signaling #43,566, LC3 type I and II) (1:500 in 3% albumin) were incubated at 4 °C overnight. After washes, cells were incubated in Texas Red conjugated secondary antibody (Jackson Laboratory, Maine USA) (1:1000 in 3% albumin) for 1 h at room temperature. Then, cells were washed and treated with YO-PRO-1 (for BCL-2 reaction, #Y3603) and TO-PRO-3 (for LC3 reaction, #T3605) (Thermo Scientific, Massachusetts, USA) for nuclei counterstaining and mounted on slides using glycerol. Immunoreaction was analyzed using a confocal microscope (LSM 510 Meta; ZEISS, Oberkocher, Germany), 10 different microscopic fields/slides; 3 slides per group, totaling 30 analyzed areas/group. Positive cells were quantified by fluorescence intensity analysis (normalized by the cell number in each image) using the ImageJ program [19 (link)].