The human dopaminergic neuroblastoma cells, SH-SY5Y subline cells were obtained from University of Heidelberg, HD, and were maintained in a humidified atmosphere containing 5% CO2. Cells were seeded in 10 mm dishes in DMEM media supplemented with 10% heat inactivated fetal bovine serum, 50 units/mL penicillin, 50 mg streptomycin, and 2 mM glutamine. After 48h of seeding the cells were exposed to freshly prepared 6OH-DA an ascorbic acid solution (10-4 M, 0.01% w/v) in cold media containing 2% serum) for 4-24h. Cells used as control were exposed to freshly prepared ascorbic acid solution at the same concentration used for the test compound. Immunocytochemistry was performed according to the method described in brief. The cells were washed and then fixed with methanol for 2 min. at-20 C. The cell were then treated to block non-specific binding. After several washing cells were incubated (15h at 4 C) with anti-Tyrosine Hydroxylase (TH)(MAB5280 mouse anti-TH) monoclonal antibodies diluted 1: 100 in 0.1% Triton X100 PBS,(Chemicon©) The next day, after washing with PBS, cells were incubated with a secondary antibody (DAKO E0433 Goat anti Mouse) diluted 1: 1000, for 1h at room. temperature. Cells were washed with PBS and then incubated with Alexa Fluor-Extravidine (Sigma) 1: 1500 in PBS for 1h. Images of immunoreactive cells were digitalized by a CCD digital camera) connected to a Nikon E800 microscope (obj. 40x) and then analyzed by a Macintosh PC with the software NIH image. Preliminary results pointed out that after 24 h of 6OH-DA treatment SH-SY5Y cells showed an increase of TH …