Prolonged reagent

Neurons were fixed at DIV 14 in cold methanol 100% for 10 min. Primary antibodies were applied in GDB buffer (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl), overnight at 4 °C. Primary antibodies used were: mouse anti-LRRK2 1:100 (clone N231B/34, NeuroMab), rabbit anti-β-3Cav2.1 1:50 (Sigma), rabbit anti-synapsin I 1:200 (Cell Signalling). Secondary antibodies were prepared in GDB buffer and used for 2 h at room temperature; secondary antibodies include: goat anti-mouse Alexa Fluor 488 (Invitrogen), goat anti-rabbit Cy3 (Jackson Immunoresearch) and Alexa Fluor 555 Phalloidin (Molecular Probes, Life Technologies). Cover slips were mounted with prolonged reagent (Life Technologies) and observed with Zeiss Observer Z1 microscope equipped with an Apotome module. The obtained images provide an axial resolution comparable to confocal microscopy^{44 (link),45 (link)}. Images were acquired with AxioObserv Z1 microscope equipped with Apotome module using a plan-Apochromat 63×/1.40 Oil objective, pixel size 0,102 μm × 0.102 μm. Colocalization studies were performed on the single plan generated by optical sectioning elaborated by Apotome module. Acquired images were analyzed with ImageJ software using the colocalization analysis plugin to detect colocalization between LRRK2 and interactors.

The autophagosome–lysosome fusion assay was performed in HeLa cells transfected with the mCherry- and GFP-tagged LC3B [23 (link)]. using PEI (jetPEI^® reagent). One day after transfection, we treated cells with trehalose (100 mM), 5a (25 μM) and 5b (10 μM) for 24 h. At the end of the treatment, we fixed cells with 4% paraformaldehyde (10 min, rt). We mounted the cover slips with prolonged reagent (Life Technologies, Carlsbad, CA, USA). Images were acquired with the optical microscope ZEISS AXIO IMAGER M2 using a plan-Apochromat 40× objective and 0.102 μm × 0.102 μm pixel size. We counted the numbers of mCherry and/or GFP-positive puncta in each cell using ImageJ.

DIV 14 neurons were fixed with 4% paraformaldehyde and 4% sucrose (10 minutes, room temperature). Cover slips were mounted with prolonged reagent (Life Technologies) and observed with Zeiss Observer Z1 microscope. Images were acquire using a plan-Apochromat 40x objective and pixel size 0,102 μm × 0,102 μm. Neuritic tree analysis was performed using NeuronJ.