Odyssey infrared imaging software

The near-infrared fluorescence assay was performed as described previously (6 (link), 27 (link)). Leftover pellets from SDS extraction were extracted in 1 M NaOH at 55°C overnight. Then, samples were dried, washed with H₂O, lyophilized, and solubilized in H₂O. Samples and standard solutions from a 10 mM oxidized dopamine stock were dropped on a Biodyne Nylon Transfer Membrane (Pall). Membranes were scanned using Odyssey infrared imaging system (LI-COR) with the 700 channel. Samples were quantified by obtaining integrated spot intensities using Odyssey infrared imaging software (LI-COR).

Oxidized dopamine analysis were performed as described by Burbulla et al. [14 (link)]. Briefly, for each experiment, 1 x 10e6 neurons were harvested and cell pellets were homogenized in 1% Triton X-100 lysis buffer. Insoluble pellets were extracted in 2% SDS/50 mM Tris by boiling and sonication. Leftover insoluble pellets from a 150,000 × g spin (30 min, 4 °C) were further extracted in 1 N NaOH, followed by incubation at 55 °C. Then, the solutions were lyophilized in a Speed Vac Concentrator until the pellet was completely dry. Pellets were washed once with ultrapure H₂O for removal of hydroxides, and then lyophilized again before the dried pellet was taken up in ultrapure H₂O and finally analyzed. About 10 mM oxidized dopamine (DA) stock was used as a standard and prepared to start from 10 mM DA (in D-PBS) mixed with 20 mM NaIO₄. Each experimental sample or standard dilution was dropped onto Nylon membranes and scanned using an Odyssey infrared imaging system. Samples were quantified by obtaining integrated spot intensities using Odyssey infrared imaging software (LI-COR).

Nitrocellulose membranes (0.45 μm, GE Healthcare, Sweden) were spotted with 1μL of monomeric or fibrillar species at 1 mg/mL (α-syn, Aβ₄₀ and lysozyme) as prepared above alongside bovine serum albumin (BSA) as a negative control. The membrane was blocked with 5% milk in Tris buffer (pH 8.0) containing 0.1% Tween 20. Primary antibody against amyloid fibril LOC (Millipore, UK) (fibril specific antibody) was diluted in TBS-T (2% milk) at 1:1000 and incubated with membrane for 1 hour. Three wash steps were performed for 10 minutes with TBS-T. Secondary antibody IRDye 800CW goat anti-rabbit IgG (H+L) (Li-Cor) was applied at 1:20000 dilution and incubated at room temperature for 1 hour. Visualization was performed using the Li-Cor Odyssey and Odyssey Infrared Imaging software at wavelength 800 nm.