Ab1903

At endpoints, cells were fixed in 4% paraformaldehyde for 15 min and washed/permeabilised with phosphate buffered saline (PBS) with 0.2% Triton X-100TM (PBS-T). Cells were incubated overnight at 4°C with primary antibodies—targeting α-syn (Mouse anti α-syn, AA 124–134, ab1903, Abcam; Mouse anti α-syn 4B12, AA 103–108, IgG1, MA1-90346, ThermoFisher; pericytes (Rabbit anti-PDGFR β, ab32570, Abcam; gt PDGFR β, AF-385, R&D systems)—diluted in immunobuffer (1% goat or donkey serum, 0.2% Triton X-100 in PBS). Cells were rewashed in PBS-T and incubated with filtered (0.22 μm syringe filter, Merck) fluorescently conjugated secondary antibodies and 20 μM Hoechst 33258 (ThermoFisher) for 2 hours at room temperature. Images were acquired using the automated fluorescence microscope ImageXpress® Micro XLS (Version 5.3.0.1, Molecular Devices) using the 20 x (0.45 NA) CFI Super Plan Fluor ELWD ADM objective lens and Lumencor Spectra X configurable light engine source. Confocal images were acquired using a LSM 800 Airyscan confocal microscope (Zeiss LSM 800 Airyscan confocal microscope) with a 40x/1.3 or 63x/1.4 NA Plan Apochromat oil immersion using the Airyscan module.

Protein was loaded on 4–12% Bis-Tris NuPAGE gels (ThermoFisher), separated by electrophoresis and transferred to Hybond PVDF membrane (GE Healthcare). For TX-100 soluble/insoluble studies, the amount of urea-SDS fraction loaded for each sample was calculated as a proportion of the protein concentration in the TX-100 soluble fraction. Because of the purity of the insoluble fraction neither β-actin nor GAPDH were detectable in the insoluble fraction. For α-syn blots, PVDF was fixed with 4% paraformaldehyde and 0.01% (v/v) glutaraldehyde for 30 min at room temperature (62 (link)). Membranes were blocked with 5% milk/PBS/0.1% (v/v) Tween 20 and incubated with the following primary antibodies: α-syn (abcam, ab1903 for mouse; ab80627 for human), α-syn phospho S129 (abcam, ab51253), β-actin (abcam, ab6276), GAPDH, (abcam, ab8245), GCase (Merck, 2E2 for human; Sigma, G4171 for mouse), GRP78/BiP (abcam, ab21685), HA (Biolegend, HA.11), histone H3 (abcam, ab1791), LIMP2 (abcam, ab16522), TFEB (abcam, ab2636), TH (abcam, ab112). Following incubation with respective HRP-conjugated secondary antibodies, blots were incubated with Immobilon Luminata Forte enhanced chemiluminescence (Merck) and images acquired and quantified using Image Lab software (BioRad). Band densities were normalized to β-actin or GAPDH.

HEK-293T neurons growing on glass coverslips were fixed in 4% paraformaldehyde for 15 min and then washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation). Cells were treated for 1 h with PBS containing 1% bovine serum albumin. To detect the expression of NR1-Rluc, cells were labeled with a mouse anti-Rluc antibody (1/100; MAB4400, Millipore, Burlington, MA, USA) and subsequently treated with Cy3-conjugated anti-mouse IgG secondary antibody (1/200; 715-166-150; Jackson ImmunoResearch, West Grove, PA, USA) (1 h each). The expression of CB₁R-YFP was detected by the YFP’s own fluorescence. The presence of α-syn fibrils was detected with a mouse monoclonal anti-human α-synuclein antibody (1/300; ab1903, Abcam). Phalloidin was detected with an AF488 pre-stained anti-phalloidin probe (1/200; A12379, ThermoFisher, Waltham, MA, USA). Nuclei were stained with Hoechst33432 (1/100 from stock 1 mg/mL; Thermo Fisher, Waltham, MA, USA). The samples were washed several times and mounted on glass slides with ShandonTM Immu-MountTM (9990402; ThermoFisher, Waltham, MA, USA). Samples were observed under a Zeiss 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with an apochromatic 63× oil-immersion objective (N.A. 1.4) and with 405 nm, 488 nm, and 561 nm laser lines.