The striatum (n = 4–6 mice/group) was dissected bilaterally at 4 °C and stored at − 80 °C until processed. As described earlier [32 (link)], tissues were homogenized in ice-cold lysis buffer, centrifuged at 14,000 rpm for 10 min and the supernatants were collected for determination of protein concentration. Aliquots of protein (20–30 μg) were subjected to 10% SDS-PAGE and transferred to Immun-Blot PVDF Membrane (Bio-Rad, Hercules, CA). Membranes were incubated in 10% dry milk in PBST solution at room temperature for 1.5 h and then in primary antibodies at 4 °C overnight. Subsequently, they were washed with PBST for 30 min and incubated in secondary anti-mouse (1:5000; Abcam ab205719) and anti-rabbit (1:5000; Abcam ab205718) antibodies at room temperature for 1.5 h. Following a rinse with PBST for 30 min, they were reacted with enhanced chemiluminescent reagent (GE Healthcare Life Sciences, Buckinghamshire, UK) and imaged with Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE). Blot intensity was quantified using ImageJ and normalized to β-actin.
Primary antibodies included rabbit anti-TH (1:500; Abcam ab112), guinea pig anti-VGLUT1 (1:1000; Millipore AB5905), mouse anti-GAD67 (1:5000; Millipore MAB5406), mouse anti-DAT (1:1000; NovusBio mAb16, Littleton, CO) and mouse anti-β-actin (1:5000; Sigma-Aldrich A5441, St. Louis, MO).
Primary antibodies included rabbit anti-TH (1:500; Abcam ab112), guinea pig anti-VGLUT1 (1:1000; Millipore AB5905), mouse anti-GAD67 (1:5000; Millipore MAB5406), mouse anti-DAT (1:1000; NovusBio mAb16, Littleton, CO) and mouse anti-β-actin (1:5000; Sigma-Aldrich A5441, St. Louis, MO).