Immunofluoresence staining
Primary neuronal cultures on poly-L-lysine coated coverslips were treated with κ-ΟR ligands for different time intervals. Cells were fixed for 10 min with 100 % methanol at -20oC and incubated overnight at 4 oC with the anti-LC3B antibody (1:200) followed by 2 h incubation with the fluorescein-conjugated secondary antibody Alexa fluor 488 goat anti-rabbit (1:100) and TO-PRO-3 (1:500) for nuclear staining. The cells were mounted on slides with Vectashield mounting media (Vector Laboratories Inc., Burlingame, CA, USA) and visualized using a Leica SP8 confocal microscope (Leica Microsystems, Germany).