Flouview3000 confocal microscope

PLA was conducted to investigate direct protein-protein interactions within cells. Approximately 20,000 cells were seeded per well in 8-well chamber slides for this experiment. Following the designated treatments, cells were stained with 50 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, USA) for 15 minutes under standard culturing conditions. After staining, cells underwent a washing step, and were then fixed with 4% PFA for 15 minutes at room temperature (RT). Subsequent steps included permeabilization in 0.2% Triton X-100 in 1X PBS for 10 minutes at RT, followed by PBS washing to remove any residual permeabilization agent. The in-situ PLA experiment was performed per the manufacturer’s guidelines, using the DuoLink kit (Sigma-Aldrich, USA). The primary antibodies used for the PLA are detailed in Supplementary Table 2. After the PLA procedure, coverslips were mounted using DAPI mounting media (Sigma-Aldrich, USA), and imaging was performed using either a Zeiss Axio Observer 7 microscope or an Olympus Flouview3000 confocal microscope.

The pCW-FLAG-α-Syn transduced SH-SY5Y cells were cultured on 8-well chamber slides. For iPSCs and NPSCs, the chamber slides were pre-coated with Matrigel and Geltrex, respectively to facilitate adherence. Fixation of the cells for IF analysis was performed by replacing the media with a 1:1 ratio mixture of fresh media and 8% paraformaldehyde (PFA) in PBS, resulting in a final concentration of 4% PFA. Post-fixation, the slides were permeabilized using 0.2% Triton X-100 in 1X PBS, followed by blocking with 5% Goat Serum-TBS-T (1× TBS with 0.1% Tween-20) to prevent non-specific antibody binding. The cells were then incubated overnight at 4°C with primary antibodies (Supplementary Table 2). Following this step, Alexa Fluor 488 (green) and 647 (red)-conjugated secondary antibodies (Thermo Fisher, USA) were incubated for 1 hour, and slides were then mounted with coverslips after applying DAPI-containing mounting media (Sigma-Aldrich, USA) to visualize the nuclei. Imaging was performed using a Zeiss Axio Observer 7 microscope or an Olympus Flouview3000 confocal microscope.