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Fluorescence Detection

LB Leah C. Beauchamp
XL Xiang M. Liu
LV Laura J. Vella
PA Paul A. Adlard
AB Ashley I. Bush
DF David I. Finkelstein
KB Kevin J. Barnham
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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.

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