Mouse anti asyn

The following antibodies were used in these studies: mouse anti-aSyn (#610787, BD Biosciences, San Jose, CA); rabbit anti-ENSA (#D5Z1U), rabbit anti-β3-tubulin (#D65A4), and AP-linked anti-mouse and anti-rabbit IgG (Cell Signaling Technology, Danvers, MA); chicken anti-MAP2 (#CPCA-MAP2, EnCor Biotechnology, Gainesville, FL); rabbit anti-TH (#AB152, Millipore, Billerica, MA); mouse anti-β-actin (#A5316 or #A5441, Sigma-Aldrich); and anti-rabbit IgG-Alexa Fluor 488 and anti-chicken IgG-Alexa Fluor 594 (Invitrogen, Carlsbad, CA).

The absolute number of extracellular NM aggregates was estimated within the same sections in which SNpc TH-positive stereological cell counts were performed (i.e. serial 5-µm-thick paraffin-embedded sections covering the entire SNpc, taking every 17th section in rats and every 6th section in mice, for a total of 6–8 sections analyzed/animal). The number of neuronophagic events were assessed in serial H&E-stained 5-µm-thick paraffin-embedded sections covering the entire SNpc. The number of p62-immunopositive Marinesco bodies, PB, and Lewy body-like inclusions was counted from SNpc sections fluorescently immunostained with guinea pig anti-p62 (1:1000, Progen), rabbit anti-ubiquitin (1:500, Dako), and mouse anti-aSyn (1:1000, BD Biosciences). The total number of p62-positive inclusions falling into each category was counted from images covering the whole SNpc region in each section. Quantifications were performed in AAV-hTyr-injected at different time-points post-AAV-hTyr injection: 0.5 m (n = 8), 1 m (n = 5), 2 m (n = 6), 4 m (n = 5), 12 m (n = 6), and 24 m (n = 5). AAV-hTyr-injected mice were analyzed at 2 m post-AAV injections: WT (n = 3), aSyn KO (n = 4). AAV-hTyr (n = 5), AAV-TFEB (n = 4), AAV-hTyr + TFEB (n = 4), and vehicle-injected rats (n = 7) were analyzed at 12 m post-injection. All quantifications were performed by an investigator blinded to the experimental groups.

Twenty-four or forty-eight hours after transfection, cells (HEK or H4) were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature (RT), followed by a permeabilization step with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at RT. After blocking in 1.5% normal goat serum (PAA, Cölbe, Germany)/DPBS for 1 hour, cells were incubated with primary antibody. Primary antibodies used were: mouse anti-ASYN (1∶1000, BD Transduction Laboratory, New Jersey, USA) or rabbit anti-ASYN (1∶1000, Abcam, Boston, USA), rabbit anti-LAMP-1 (1∶1000, Abcam, Boston, USA), anti-Giantin (1∶1000, Abcam, Boston, USA) for 3 hours or overnight and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG and/or Alexa Fluor 555 goat anti rabbit IgG, (Life Technologies- Invitrogen, Carlsbad, CA, USA) for 2 hours at RT. Finally, cells were stained with Hoechst 33258 (Life Technologies- Invitrogen, Carlsbad, CA, USA) (1∶5000 in DPBS) for 5 minutes, and maintained in PBS for epifluorescence microscopy.

Mice were anaesthetised with pentobarbitone 20% (w/v) (100 μL or 20 mg per mouse, i.p.) and transcardially perfused with PBS (pH 7.4), followed by 4% paraformaldehyde (PFA) (v/v). Brains were post-fixed in 4% PFA for 24 h, cryoprotected in 30% sucrose for 72 h before 35 μm sections were cut. Free-floating sections were stored at -20°C in anti-freeze solution (50% PBS, 25% ethylene glycol, 25% glycerol) until staining was performed. Sections were washed in PBS, blocked in 10% normal goat serum for 1 h at room temperature and incubated in primary antibodies diluted in blocking solution overnight at 4°C. The primary antibodies used were mouse anti a-syn (1:500, BD Biosciences), and rabbit anti-tyrosine hydroxylase (TH) (1:500, Millipore). The next day, sections were washed in PBS containing 0.1% Triton-X100 (PBS-TX) and incubated with appropriate Alexa fluor goat anti-mouse 488 nm and/ or goat anti-rabbit 594 nm secondary antibodies diluted in PBS-T (1:200, Life Technologies) for 1h at room temperature. For nuclear staining, sections were incubated in 4’,6-diamidino-2-phenylindole (DAPI) (1:2000, Life Technologies) for 10 min at room temperature. Sections were mounted with FluorSave mounting medium (Calbiochem) before being visualised with an Evos FL auto imaging system (Life Technologies).