Blocker fl fluorescent blocking buffer

Membranes (0.45 µm nitrocellulose, BioRad, USA) were blocked in Blocker™ FL Fluorescent Blocking Buffer (ThermoFischer, Aus) for 1 h at RT. All antibodies (α-syn (1:5000), BD biosciences; β-actin (1:10,000), CST; synaptophysin (1:5000), CST) were diluted in TBS-T. Membranes were washed in TBS-T for 21 min (3 × 7 min) before and after incubation with fluorescent secondary antibodies (Licor, Aus). Proteins were visualized with the Licor Odyssey fc system (Licor, Aus) and analyzed via signal intensities (ImageStudio 5.2, Licor, Aus). Samples normalized to β-actin, except for 4-HNE which was normalized to total protein as determined by Revert Total Protein Staining (Licor, Aus) according to manufacturer’s direction.

Fluorescent western blots were performed as previously published (19). Samples lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X‐100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) were loaded in Laemmli buffer in 5% β‐mercaptoethanol and separated by electrophoresis with Bio‐Rad Criterion TGX Stain‐Free Precast Gel (4%–20%; Bio‐rad, 5,678,095) in a Bio‐Rad Criterion Cell, before transfer with a Criterion Blotter to Immobilon‐FL PVDF 0.45 um (Merck, IPFL00010) membrane. Immunoblots were blocked with Blocker FL Fluorescent Blocking Buffer (37565, Thermo Fisher), incubated with primary anti‐GFAP rabbit antibody (AB5804, Sigma‐Aldrich), for 16 h at 4°C (1:2000), and secondary anti‐rabbit Alexa Fluor Plus 647 antibody (A32795, Thermo Fisher) for 1 h (1:5000), facilitated with an automated staining system GOBlot (Cytoskeleton). Fluorescent immunoblots were visualized with a ChemiDoc MP (Bio‐Rad; Figure S10).