Mouse anti α synuclein

Cells were cultured on a round slide. After treatment, the slide was washed with 0.01 M PBS and fixed with 4% PFA as previously described (34 (link)). The primary antibody (Mouse anti-α-synuclein: BD, USA; Rabbit anti-NeuN: Abcam, USA) was added and incubated with the cells for 48 h at 4°C. Then, the slide was exposed to the Alexa Fluor^® antibody (Invitrogen, USA) and incubated for 1 h at 37°C. The nucleic acids were stained with DAPI (Invitrogen, USA). Following a final wash and mounting with anti-fade medium (Sigma, USA), images were acquired using a fluorescence microscope (Nikon, Japan). The fluorescence intensity was determined using Image-Pro Plus, Version 6.0 (MediaCybernetics, Inc., USA).

Directly after image acquisition at the microscope, cells were washed twice with sterile PBS and then lysed on ice for 10 min in lysis buffer as previously described^{17 (link)} or RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 0.5% DOC, 0.1% SDS, complete protease inhibitor cocktail mix, Roche). Lysates were centrifuged at 13 000 r.p.m. for 10 min at 4 °C. Supernatants were collected and protein concentration was determined by the Bradford method. Samples were stored at −80 °C until used. 10 to 30 μg of protein per slot were separated on a 12% SDS polyacrylamide gel and transferred to a polvinylidene difluoride membrane. Membranes were incubated for 1 h in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween, pH 7.5) containing 5% milk to saturate all non-specific binding sites. Incubation with primary antibodies was overnight at 4 °C in 1% milk+TBST (1 : 1500 goat anti-DJ-1, Santa Cruz Biotechnology, Dallas, TX, USA; #Sc-27006; 1 : 3000 mouse anti-α-Synuclein, BD #610787; 1 : 5000 mouse anti-β-actin, Sigma-Aldrich, St. Louis, MO, USA; #A5441). Blots were developed using horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-goat 1 : 10 000 Jackson Immunoresearch, West Grove, PA, USA; #705-035-003, anti-mouse 1 : 10 000 Sigma #50185-ILM-F) and the Immobilon chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).

Wt C57Bl/6 mice (1–3 months old, female) were anesthetized with ketamin/xylazin and perfused with PBS. After incubation of brains with 30 % sucrose for 24 h at 4 °C, brain tissue was embedded in TissueTek®O.C.T (Sakura, Alphen a.d.R., Nehterland), frozen at −80 °C and cut in 12 μm sections using a cryostat. Sections were fixed with 2 % PFA for 30 min, permeabilized with 0,5 % saponine for 10 min and treated with 3 % H₂O₂ for 10 min, followed by blocking with Roti®-ImmunoBlock (Roth, Karlsruhe, Germany) for 1 h. Samples were co-immunostained with primary antibodies mouse-anti-α-synuclein (1:200, syn-1,BD,New Jersey, USA) and rabbit-anti-SOD1 (1:100, ADI-SOD100, Enzo life science, New York, USA) in Roti®-ImmunoBlock for 1 h 45 min at RT. After washing with PBS, sections were blocked with 5 % goat-serum in PBS for 15 min and incubated with fluorophore conjugated secondary antibodies (1:750, goat-anti-rabbit-Alexa546 and goat-anti-mouse-Alexa-488, both Life technology, Carlsbad, USA) in 5 % goat serum for 1 h. Then sections were washed with PBS, incubated with xylol for 2 min and 100 % ethanol for 3 min and coverslipped using DAPI Fluoromont®G (SouthernBioTech, Birmingham, USA). To avoid unspecific binding of secondary antibodies, sections were also single stained with either α-synuclein or SOD1 primary antibody and both secondary antibodies.