Mabn389

For the detection of fluorescent proteins after infection, free-floating sections were washed three times in phosphate-buffered saline (PBS), blocked in PBS containing 4.5% bovine saline albumin (BSA) for 1 h, then incubated overnight at 4°C in PBS containing 3% BSA, 0.2% Triton X-100, and one of the following antibodies of interest. Sections were rinsed three times in PBS and incubated with Alexa Fluor 488-labeled antimouse or rabbit IgG or Alexa Fluor 555-labeled antimouse or rabbit IgG (Invitrogen) at room temperature for 1 h. The sections were rinsed in PBS and the nuclei were counterstained with DAPI (dilution 1:10,000, Wako) for 3 min. The sections were mounted in Mowiol.
Series of sections were stained for aSyn to localize the injection site in aSyn-injected rats. Tyrosine hydroxylase (TH) (Rabbit, Millipore, ab152) immunostaining was used in order to quantify the loss of dopaminergic neurons in the injected rats. The following antibodies were used to detect specific synucleins: N-terminal rat aSyn (Mouse, Bd Transduction Laboratories, 610787), recognized amino acid 121–125 of human aSyn (Mouse, Santa-Cruz, SC-12767), pS129 aSyn (Rabbit, Abcam, ab51253), and aggregated forms of aSyn (Mouse, Millipore, clone 5G4, MABN389).

WT aSN monomers and purified αSOs were diluted in PBS. 2 μL containing 60 ng sample or buffer was spotted on a nitrocellulose membrane (Cell signalling, Danvers, MA) and air-dried for 30 min at RT. Blots were blocked for 1 h in Odyssey blocking buffer in PBS (LI-COR, Lincoln, NE) and incubated O/N at 4°C with primary antibody diluted in blocking buffer containing 0.1% Tween-20 (Sigma). The following antibody dilutions were used: mouse anti-aSN 211, 1:1000 (sc12767; Santa Cruz biotechnology, Santa Cruz, CA); mouse anti-aggregated aSN 5G4, 1:500 (MABN389, Millipore, Billerica, MA); rabbit anti oligomer A11, 1:500 (AHB0052, Invitrogen, Carlsbad, CA). Subsequent blots were washed 3x 5 min at RT with PBS-Tween 0.1% and incubated for 1 h at RT with the appropriate infrared secondary antibody, IRDye 800CW (LI-COR, Lincoln, NE) diluted 1:5000 in blocking buffer added with 0.1% Tween-20. Blots were washed 3x for 5 min at RT with PBS-Tween 0.1% and 2x for 5 min at RT with PBS, before visualizing using an Odyssey classic infrared imaging system (LI-COR, Lincoln, NE). Images were processed using Image studio lite software (LI-COR, Lincoln, NE, V5.0). The experiment was carried out in triplicate.