*4.6. Immunocytochemistry*

Immunocytochemistry was performed and modified according to Iida's study. Briefly, SH-SY5Y cells were seeded in 12-wells plates and incubation with MPP<sup>+</sup> and different reagen<sup>t</sup> for 24 h. After washing with PBS for three times and fixing with PBS containing 4% (wt/vol) paraformaldehyde for 15 min and then permeabilized with 0.5% (wt/vol) Triton X-100 and blocked with 5% goa<sup>t</sup> serum for 1 h at room temperature. Subsequently, the cells were incubated with rabbit monoclonal anti-Akt (1:200), anti-PI3K (1:200), anti-Bax (1:100), anti-Bcl-2 (1:100), anti-GSK3β (1:200), anti-pPI3K (1:200), anti-p53 (1:100), anti-NGF (1:200), anti-pAkt (1:200) antibodies at 4 ◦C overnight. After washing with PBST and incubated with the second antibody (1:200) in PBST for 1 h. After the samples were washed with PBS three times, they were embedded in DAPI for 5 min and then washed with PBST 4 times. The images were obtained using an Olyba microscope. The mean fluorescence intensity was calculated by Image-Pro (Rockville, MD, USA).
