Ab184451

On the day following the PET scan, the rats under anesthesia state were perfused intracardially with cold 4% paraformaldehyde. According the routine immunohistochemistry procedure as previously reported [29 (link)], the primary antibodies of anti-TH (1 : 1000; ab184451, Abcam) and anti-DAT (1 : 300; ab184451, Abcam) were used in the current study. The number of TH/DAT-positive cells in the SN was counted, and the integrated density of DAT-positive fibers in Str was assessed with the Imaging J threshold plugin (Image J, NIH/USA) for each hemisphere.
Immunofluorescent staining was performed with the primary antibodies against mGluR5 (1 : 200, ab76316, Abcam), TSPO (1 : 100, ab154878, Abcam), ionized calcium–binding adaptor molecule-1 (Iba-1) for microglia cells (1 : 100, Ab5076, Abcam), and DAPI for nuclei (1 : 5000, sc-3598, Santa Cruz). The mGluR5 positive or TSPO positive microglia cell numbers in SN per square millimeter were counted separately. The ratio of the lesioned (left) to the intact (right) side was analyzed and utilized for the further statistical analysis of both the immunohistochemical and immunofluorescent staining.

Rat anti-DAT (MAB369; Millipore; 1:2000 dilution) or rabbit monoclonal anti-DAT (AB184451, Abcam, 1:1000 dilution for Fig. S2, A and B only), rabbit anti-TH (AB152, Millipore, 1:10,000 dilution), rabbit anti-pSer40 TH (AB5935, Millipore, 1:5000 dilution), mouse antitransferrin receptor (clone H68.4, Thermo Fisher; 13-6800). Secondary antibodies conjugated to horseradish peroxidase were all from Jackson ImmunoResearch, and immunoreactive bands were visualized by chemiluminescence using SuperSignal West Dura (Thermo Scientific). Nonsaturating immunoreactive bands were detected using either a VersaDoc 5000MP or a ChemiDoc imaging station (Bio-Rad) and were quantified using Quantity One software (Bio-Rad). Surface DAT was calculated by normalizing the biotinylated DAT signal to its corresponding amount of total DAT in that sample, detected in parallel from the same exposure of the same immunoblot. Raw surface DAT values are expressed as %total. For most experiments, DAT surface values following drug treatment were normalized to vehicle-treated samples, obtained from the contralateral hemislice in parallel. Surface DAT bands, and their corresponding total lysate, shown for each experiment were taken from the same exposure of the same immunoblot, and brightness/contrast levels were set identically for all blots. Boxed bands were cropped and rearranged for presentation purposes only.