Proximity Ligation Assay (PLA)

The PLA assay was performed as described previously with minor modifications ⁴⁸. The following antibodies were used for the PLA experiments: Syn 204 against h-αSyn (Santa Cruz Biotechnology Cat# sc-32280, RRID:AB_628319)(1:100) and EPR12790 against VAMP-2 (Abcam Cat#

ab214590)(1:300).

The in-situ PLA was performed on fixed primary neurons

with DuoLink PLA technology probes and reagents (Sigma-Aldrich Cat# DUO92002,

DUO92004, DUO82049, DUO92008, and DUO92014), following the manufacturer’s

protocol. First, the neurons were permeabilized with PBS + 0.4% Triton X-100

for 10 min.

After two PBS washes, the cells were incubated with a blocking

solution for 2 hours at 37 °C and then incubated with the primary antibodies

for 30 min at room temperature.

The coverslips were washed twice for 5 min with

buffer A, followed by incubation with the PLA probes (secondary antibodies

against two different species bound to two oligonucleotides: anti-mouse MINUS (Sigma-Aldrich Cat#

DUO92004 (also DUO92004-30RXN, DUO92004-100RXN), RRID:AB_2713942) and anti-rabbit PLUS) (Bethyl Cat# OLK-92002-0100,

RRID:AB_10950581)in antibody diluent for 30 min at 37 °C. After two washes of 5

min with buffer A, the ligation step was performed with ligase diluted in

ligation stock for 30 min at 37 °C.

The coverslips were washed with buffer A

twice for 2 min before incubation for 50 min with amplification stock solution

at 37 °C. After two washes of 10 min with buffer B. Finally, the coverslips

were washed with PBS and mounted with Duoli in situ tu mounting medium

(Sigma-Aldrich Cat# DUO82040-5 ML).

A negative control experiment was performed

for every antibody, where only one antibody was incubated with the PLA probes.

The experiments were performed 2 times. The experiments were performed 2 times.

Average signal intensities were measured using the  MetaMorph Microscopy Automation and Image

Analysis Software (RRID:SCR_00236 https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref) https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#grefgref) and plotted using GraphPad Prism

software [(RRID:SCR_002798) http://www.graphpad.com/].