Alexa fluor 488 or alexa fluor 594 conjugated secondary antibody

Animals were perfusion-fixed with 4% paraformaldehyde (PFA, in 0.1 M phosphate buffer, pH 7.3), and brains were removed, cryoprotected, dissected into 40-μm-thick coronal sections, and subjected to chromogenic immunohistochemistry, as previously described (63 (link)). A Zeiss Axio Imager M2 light microscope with color charge-coupled device (CCD) camera (AxioCam, Zeiss) was used to capture all images.
Immunofluorescence staining was also conducted as previously described (63 (link)). Briefly, sections were blocked in 10% normal goat serum (Invitrogen), and sequentially incubated with primary antibodies (for 48 h) and Alexa Fluor-488- or Alexa Fluor-594-conjugated secondary antibodies (Life Technologies). Sections were mounted on glass slides, coverslipped in mounting medium (Prolong containing DAPI, Invitrogen), and images were acquired on a Nikon A1plus-RSi scanning confocal microscope. For primary neurons, fixed cells were incubated with primary antibodies to FLAG-M2 (Sigma) and either pS1292-LRRK2 (MJFR-19-7-8, Abcam) or pT73-Rab10 (MJF-R21-22-5, Abcam), and anti-mouse IgG-AlexaFluor-488- and anti-rabbit IgG-AlexaFluor-546 secondary antibodies. Neurons were mounted and imaged as above.

For imaging, 4 × 10⁴ HeLa cells were seeded in chamber slides (Lab-Tek chambered coverglass; Thermo Fisher Scientific) and transfected 24 h later with 0.3 µg of any indicated DNA with X-tremeGENE 9 (Roche). After treatment, cells were fixed with 4% (vol/vol) paraformaldehyde (Electron Microscopy Services) in PBS for 20 min at room temperature and washed three times in PBS. Cells were permeabilized in 0.5% Triton X-100 in PBS and stained with the primary antibodies as follows: mouse monoclonal anti-Parkin (catalog no. sc-32282; Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Tom20 (catalog no. sc-11415; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-ubiquitin (catalog no. MAB1510; EMD Millipore), and mouse monoclonal anti-HA (catalog no. MMS-101R; Covance) in 5% (wt/vol) BSA (Thermo Fisher Scientific) for 18 h at 4°C, followed by anti–mouse or anti–rabbit Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies (Life Technologies). For Parkin translocation, counts of Ser/Thr mutant samples were manually counted for translocation phenotype (50 cells/mutant in each of two independent replicates). All images were acquired using LSM software (Carl Zeiss) with fixed cells in PBS at room temperature on an inverted confocal microscope (LSM510 Meta; Carl Zeiss) using a 63×/1.4 NA oil immersion Plan Apochromat objective.

Immunocytochemistry was performed as described previously (21 (link)). Briefly, the cultured cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, USA) for 10 min, followed by washing with DPBS. Next, the cells were blocked and permeabilized with 3% bovine serum albumin (BSA, Thermo Fisher Scientific) and 0.3% Triton X-100 (Sigma-Aldrich) in DPBS for 1 h at room temperature. All samples were then incubated with primary antibody solution overnight at 4°C. The next day, after washes with 0.1% BSA in DPBS, samples were incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Images were captured using a Fluoview FV1000 confocal microscope (Olympus, Japan). The antibodies used in this experiment are listed in Supplementary Table S2.