Sc 14002

Immunohistochemistry (IHC) was conducted as described by Campolo et al. [49 (link)] and briefly reported below. The sections underwent an overnight incubation with primary antibodies, namely anti-DAT (1:100; sc-14002; Santa Cruz Biotechnology) and anti-α-synuclein (1:100; sc-7011; Santa Cruz Biotechnology), subsequent to the paraffin being removed using a declining scale of alcohols. Using the VECTASTAIN Universal Quick Kit, Peroxidase, R.T.U. (PK-7800; Vector Laboratories, Burlingame, CA, USA), the sections were then meticulously cleaned with PBS before being probed with the secondary antibody. The reaction was observed using the water-soluble chromogenic substrate 3,3′-Diaminobenzidine (DAB), with Nuclear Fast Red serving as a counterstain. The percentage area of immunoreactivity was examined using a computerized image analysis system, and it was determined by counting the number of positive pixels. The proportion of total tissue area (brown staining) was expressed as a percentage among five random fields at a 40× magnification. For the analysis, a Nikon Eclipse Ci-L microscope was used. Figures are shown at 40× magnification.

The in situ PLA allows the detection of protein-protein interactions in situ in intact tissues [7 (link),27 (link),30 (link),31 (link),32 (link),33 (link),48 (link)]. For the fluorescence human in situ PLA studies, we analyzed paraffin embedded brain sections from the caudate putamen of PD patients and age-matched controls by using the Duolink assay kit (O-Link Bioscience, Uppsala, Sweden) with a protocol adapted from the manufacturer’s instructions. Briefly, following deparaffinization and antigen retrieval with 10 mM sodium citrate buffer, sections were incubated in a blocking solution (provided by the kit) for 1 h at rt and then with the primary antibodies recognizing DAT (sc-14002, Santa Cruz Biotechnology), and α-synuclein (MA5-12272, Syn211, Thermo Fisher Scientific) at 1:100 dilution at 4 °C. On the following day, samples were washed and then incubated with a PLA probe solution for 1 h at rt. Sections were then washed and incubated with the ligation solution for 45 min at 37 °C, and then with the amplification solution at 37 °C for 100 min. Finally, cell nuclei were then counterstained with Hoechst 33258 (Sigma-Aldrich), and sections were mounted using the Vectashield mounting medium (Vector Laboratories).

MN9D cells were seeded on a six-well chamber slide for treatment. The cells were then rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at 26–28 °C. The cells were rinsed with PBS, then blocked and permeabilized with 1% normal goat serum and 0.5% TritonX-100 in PBS. The cells were then incubated with the following primary antibodies: mouse monoclonal anti-DYT5b antibody (1:200 dilution in blocking buffer, sc-25269, Santa Cruz Biotechnology), or rabbit polyclonal anti-DAT antibody (1:200 dilution in blocking buffer, sc-14002, Santa Cruz Biotechnology) overnight at 4 °C. The following day after three washes with PBS, the cells were incubated with appropriate secondary antibodies: DyLight 488 anti-rabbit IgG and DyLight 594 anti-mouse IgG (1:200, DI-1088, DI-2594, both Vector Labs, Burlingame, CA, USA) for 40 min at 26–28 °C. The cell nucleus was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (C0065, Solarbio, Beijing, China) for 5 min. After three washes with PBS, the cover slips were sealed with Antifade Mounting Medium (P0128, Beyotime Biotechnology). The results were arranged and analyzed with Image-Pro Plus 6.