The dorsal striatum was dissected from the fresh brain slices made with a microslicer (VT 1000S, Leica Microsystem, Wetzlar, Germany) in artificial cerebrospinal fluid containing (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), MgCl2 (1.3), CaCl2 (2.4), glucose (10), and NaHCO3 (26), frozen and stored at −80 °C until processed. The samples were sonicated in 1% sodium dodecyl sulfate (SDS) and boiled for 10 min. The 1 % SDS was diluted in water from 10% SDS (prepared with 18 MΩ water, Bio-Rad, Cat. No. 1610416, Hercules, CA, USA). Protein concentration was determined in each sample with a bicinchoninic acid protein assay (BCA-kit, Pierce, Rockford, IL, USA, Cat. No. 23225). Equal amounts of protein (30 μg) were re-suspended in the sample buffer (4 × Laemmli Sample Buffer, Bio-Rad, Hercules, CA, USA, Cat. No. 1610747, added with 10 % β-mercaptoethanol, Sigma, St Louis, MO, USA, Cat. No. M3148) and separated by SDS–polyacrylamide gel electrophoresis using a 9 % acrylamide gel (Acrylamide/Bis-acrylamide, 30% solution: Sigma, St Louis, MO, USA, Cat. No. A3699) and transferred to a nitrocellulose transfer membrane (Bio-Rad, Hercules, CA, USA, Cat. No. 1620115). We performed Western blotting in duplicates for the samples from aged mice (20–21 months), as performed in previous studies [18], due to the low availability of mice in this group. The membranes were incubated for 1 h at room temperature with 5% (w/v) fat-free dry milk (Cell Signaling, Danvers, MA, USA, Cat. No. 9999S) in TBS-T (Tris base 0.05 mol/L, NaCl 0.15 mol/L, tween 0.1%). Immunoblotting was carried out with primary antibodies in 5% dry milk dissolved in TBS-T at 4 °C overnight. Antibodies were obtained from Sigma-Aldrich, St Louis, MO, USA (tyrosine hydroxylase (TH), Cat. No. T2928, dilution 1:2000; β-actin, Cat. No. A2228, dilution 1:2000) and Millipore, Temecula, CA, USA (DAT, Cat. No. MAB369, dilution 1:1000). The membranes were washed three times with TBS-T and incubated for 1 h at room temperature, with secondary horseradish peroxidase-linked Anti-Rat IgG (H + L) (Thermo Scientific, Rockford, IL, USA, Cat. No. 31470, 1:5000 dilution) or Anti-Mouse IgG (H + L) (Thermo Scientific, Rockford, IL, USA, Cat. No. 32230, 1:5000 dilution). At the end of the incubation, membranes were washed six times with TBS-T, and immunoreactive bands were detected by enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA, Cat. No. 170-5061). The membranes were then scanned in ChemiDoc MP system (Bio-Rad, Hercules, CA, USA) and quantified with ImageJ 1.50b software (NIH, Bethesda, MD, USA). The protein amounts were normalized to the value of β-actin and expressed as a percentage of the averaged value obtained for WT mice.