Cerebral cortex samples were collected, dissolved and proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% polyacrylamide gels. Proteins were transferred onto Immobilon nitrocellulose membranes. The membranes were incubated with primary antibodies for 1 hour at room temperature and washed with Tris-buffered saline + 5% Tween-20 (TBST). The following primary antibodies were used: a rabbit polyclonal antibody for anti-LC-3 (1:2000, ab48394; Abcam), a rabbit polyclonal antibody for anti caspase-3 (cell apoptosis marker; 1:300, ab4051; Abcam), and a rabbit polyclonal antibody for anti Beclin-1 (1:3000, ab62557; Abcam). After washing in TBST again, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 1.5 hours at room temperature. Following two rinses and four washes with TBST, the optical density results were quantified by Quantity 4.5 Software (Bio-Rad Laboratories, Hercules, CA, USA) and ImageJ (NIH, Bethesda, MD, USA). GAPDH (1:1000, ab8245; Abcam) was used as internal standards.