). For generating α-syn stable cell line, α-syn DNA was cloned into the pLX208 plasmid with a hygromycin selection marker. 2.5 µg of these lentiviral DNA were then mixed with 0.25 µg pVSVG and 2.25 µg ∆8.9 lentiviral helper plasmid in 250 µl of DMEM. Then, the DNA mix was incubated with 25 µL PEI max solution at RT for at least 10 min. The DNA and PEI mix was finally added to 70–90% confluent HEK293T cells in the T25 flask.
Confocal imaging was performed on a Nikon Eclipse Ti2 inverted confocal microscope with 60X oil-immersion objectives, outfitted with Yokogawa CSU-X1 5000RPM spinning disk confocal head and ORCA-Flash 4.0 LT + sCMOS camera. The following combinations of laser excitation and emission filters were used for corresponding fluorophores: DAPI , EGFP , Alexa Fluor 568 .
Immunofluorescence analysis of α-syn PFF-treated primary cortical neuron culture and α-syn PFF-injected brain section Primary cortical neurons were fixed with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100. The cells were incubated overnight at 4 °C with rabbit anti-α-syn antibody as primary antibody for α-syn pathology assay or mouse anti-NeuN antibody for neurotoxicity, then incubation with anti-rabbit IgG secondary antibodies conjugated to Alexa-fluor 568 and Hoechst for 1 h at RT. For brain sections, 40 μm thick brain sections were blocked in with 10% goat serum with 0.
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