Collect confocal z-stack images from fluorescent-labeled tissue samples. Here we used DHE-treated mice tissue counter-stained with human alpha-synuclein or tissue co-immunolabeled with a human alpha-synuclein and SynO2 antibodies.

Create a 3D surface rendering model of h-alpha-synuclein–immunoreactive

DMnX neurons using the Imaris software.

By applying a constant intensity threshold select ox-DHE puncta and filter through h-alpha-synuclein–immunoreactive neuronal surfaces. This will allow for specific detection of ox-DHE puncta within immunoreactive

neurons

Quantify puncta on a per-cell basis using the quantification tools.

Generate 2D images that from 5-thick z-stack images using maximum

intensity projection function of the Zen software (Carl Zeiss).

Select human alpha-synuclein labeled neurons by applying a 120µm²

size exclusion filter

Measure Syn-O2 intensity within these cells using the measure tool.