Darpp32

Brains were serially sectioned at 30 μm using a Cryotome FSE and placed into a 12 well plate filled with PBS with about 14 sections per well. The wells were put in 10% SEA BLOCK (Thermo) for an hour on an orbital shaker at 60 rpm. Sections were moved into primary solution: mouse anti‐rabbit MAP2 (1:1000, Abcam) and rabbit anti‐mouse DARPP32 (1:1000, Abcam) and placed on a 4°C orbital shaker overnight. Sections were then washed with PBS three times and placed into secondary antibody stain consisting of 5% SEA BLOCK, AlexaFluor 488 anti‐mouse (1:1000, LifeTech), and AlexFluor 647 anti‐rabbit (1:1000, LifeTech) on a 4°C orbital shaker for an hour. After incubation, the wells were spiked with Hoescht (1:1000) for 5‐10 minutes on an orbital shaker, then transferred to phosphate buffered saline plus 1% Triton‐X (PBST) and washed three times. To utilize the autodetection software on the AxioScan, sections were immersed in 0.01% Sudan Black in 70% EtOH, gently agitated for 1 minute and then allowed to rest for 1 minute before being transferred into fresh PBS. The sections were then mounted onto microscope slides (Thermo Fisher Scientific) sequentially from the beginning to the end of the striatum and covered with a coverslip using Fluoromount (Sigma). Whole brain sections were then scanned and stitched together at 20× using a Zeiss AxioScan.

Blocked membranes were incubated with the primary antibody, which was rabbit monoclonal total DARPP-32 (1:50,000; Abcam), rabbit monoclonal pDARPP-32 Thr34 (1:1000; Cell Signaling Technology), or rabbit polyclonal pDARPP-32 Thr75 (1:1000; Cell Signaling Technology). After washing three times with TTBS, the primary antibodies were detected using peroxidase-labeled goat anti-rabbit IgG (H + L) antibodies (1:5000; Vector Laboratories) for 2 h at room temperature (25 °C).

Neural stem cell and noradrenergic neural cell identity was confirmed by PCR-based detection of neural stem cell (NSC) gene markers: Sox2, Gbx2, Cd-81, Cdh1, S100b, Dach1, Pax6, Olig1, or neural differentiation markers: Cspg4, DβH, Darpp32, Nestin. Moreover the neurospheres were immunostained for neural stem cell markers: Nestin (1: 500; DSHB, Iowa, USA^{90 (link)}), Foxg-1 (1:100; Abcam, Cambridge, UK), Emx1 (1: 100; Millipore, Burlington, USA) and Emx2 (1:100; Abgent, San Diego, USA) and differentiated neurons for Th (1:100; Abcam), S100b (1:100; Abcam), DβH (1:500; Abcam), Darpp32 (1:50; Abcam).

The total amount of protein in the lysates was determined by BCA protein assay (AMRESCO). Samples (50 μg protein) were separated by 10% SDS-PAGE and electroblotted to PVDF membranes, which were blocked by incubation in 5% non-fat dry milk dissolved in TBS-T (150 mM NaCl, 50 mM Tris, 0.05% Tween 20). Following transfer, proteins were probed using the following primary antibodies: D₁R, DARPP32, GABA_ARβ1, GABA_ARβ3, TrkB, pAKT, AKT, synaptotagmin, synapsin I, PSD95, spinophilin, and β-actin (Abcam). Then, horseradish peroxidase-conjugated secondary antibody was used. After extensive washing, protein bands detected by antibodies were visualized by ECL reagent (Thermo) after exposure on Kodak BioMax film (Kodak).