Ab5084p

The quantitative analysis of D2 receptor expressions in the striatum was assessed by western blotting. Tissues were lysated in 10 mM Tris–HCl pH 8, 150 mM NaCl, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 0.1% w/v sodium dodecyl sulfate, 1 mM EDTA, 5 mM β-mercaptoethanol, 1 mM PMSF, and protease inhibitor cocktail (Sigma-Aldrich). Thirty µg of proteins were loaded on a 9% SDS polyacrylamide gel and subjected to electrophoresis under reducing condition. The proteins were then transferred to a nitrocellulose membrane (Bio-Rad). The blots were incubated overnight at 4 °C with a rabbit polyclonal anti-D2 receptor antibody (1:1000, AB5084P; Millipore); or mouse anti-β-actin (1:10,000; Sigma-Aldrich) as a reference standard. Immune-reactive bands were revealed by horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch), incubated in a lumi-light-enhanced chemiluminescence substrate (Bio-Rad) and exposed to chemidoc (Bio-Rad). Densitometric analysis of scanned blots was performed using the NIH ImageJ version l.29 program (NIH, Bethesda, MD, USA).

GBM cells were plated in TIC media at a density of 250,000 cells per dish in T25 tissue culture flasks and cells collected after 24 hours. For immunoblotting, cells were lysed in RIPA buffer (Thermo Scientific 89901) supplemented to 2% SDS and proteins were separated to determine basal expression of Dopamine receptors 2, 3 and 4. Antibodies included those against Dopamine Receptor D2 (AB5084P, Millipore), Dopamine Receptor D3 (ab42114, Abcam), and Dopamine Receptor D4 (pAb-324405, Sigma).

Immunohistochemical staining to determine levels of DAT and D₂R proteins and TH activity was conducted with striatal slices. To prepare for staining, the slices were prewashed in PBS (0.01 M, pH 7.4) three times for 5 min each, then sequentially treated with 0.2% Triton X-100 in PBS for 5 min and with 0.3% H₂O₂ in PBS for 10 min, and washed in PBS three times for 5 min each, all at room temperature. Slices were initially incubated with 10% normal goat serum for 10 min (or normal donkey serum for TH measurement), then incubated for 20 h at 4°C with the primary antibodies (D₂R antibody AB5084P, Millipore, CA, USA, 1:200 dilution; DAT antibody MAB369, Millipore, CA, USA, 1:100 dilution; TH antibody T1299, Sigma, CA, USA, 1:10,000 dilution), washed three times in PBS, and incubated for 60 min at 37°C with the secondary antibodies (anti-rabbit antibody PV-6001, ZSGB-BIO, CA, USA; anti-rat antibody ZB-2307, ZSGB-BIO, CA, USA; anti-mouse antibody PK4002, Vector, CA, USA). Slices were then washed five times for 3 min each in PBS, and incubated in 100 μL of 3,3′-diaminobenzidine tertrahydrochloride (DAB) for 3 min. The slices immunostained for TH were incubated in the ABC (VECTASTAIN ABC kit, ZSGB-BIO, Beijing, China) reagent for 30 min at 37°C and washed five times for 3 min each in PBS prior to the DAB treatment. Final wash of the slices was done in distilled water.

For experiments of D1R and D2R colocalization, we eliminated any use of secondary antibodies by directly conjugating the anti-D1R antibody (Sigma, D2944) to Alexa Fluor-488, and the anti-D2R antibody (Millipore, AB5084P) to Alexa Fluor-568, accordingly to the manufacturer's instructions (Invitrogen). Coronal sections (16 μm, Bregma:1.6 ± 0.6) from perfused rat brain were blocked (1% BSA, 0.1% Triton-X in 0.05M PBS) and then incubated for 96 h at 4°C with anti-D1-488 (1:200) and anti-D2-568 (1:200). After three washes, the last one containing DAPI, the slides were mounted (Dako), and the images were acquired in sequential mode in the same conditions described above.