For immunostaining, DIV17-21 neurons were fixed in 100% ice-cold methanol solution

for 15 min at room temperature, followed by extraction in 0.1 % Triton X-100

for 10 min, blocked in 10% BSA for 1 h at room temperature, and incubated

overnight at 4°C

with primary antibodies. We found that the methanol fixation was better than

paraformaldehyde fixation for pSer129 α-syn staining.

After washing with 1x PBS, neurons were blocked again for 30 min at room temperature and incubated with secondary antibodies (Alexa Fluor antibodies from Invitrogen

(RRID:AB_2633275, RRID:AB_2762824,RRID:AB_2633282) (1:500)

for 1 h at room temperature.

Images were acquired at 40X magnification. Z-stack

images were obtained as previously described , and all images were acquired and

processed using the MetaMorph

Microscopy Automation and Image Analysis Software (RRID:SCR_00236 http://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref) http://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#grefref). 

For α-syn Ser129 and VAMP2 colocalization analysis in cultured neurons, 5-6 regions

of interest (ROIs) of 200x200 pixels were placed on each image. A total of 26

ROIs were used for this analysis.

Object-based colocalization analysis was done

using MATLAB (RRID:SCR_001622)( http://www.mathworks.com/products/matlab/). First, automatic puncta detection was done using local maxima. Next, detected puncta are said to have co-localized if the distance between their centers is less than the maximum radius of the two

particles.