Brains from male WT SynGFP [61 (
link)], E46K SynGFP and A53T SynGFP that were 3–4 months of age were dissected immediately post mortem and olfactory bulbs and cerebellum removed. Remaining brain tissue was homogenized in ice-cold lysis buffer (50 mm Tris–HCl, pH 7.5, 5 M guanidinium, protease inhibitor mixture (Roche Complete)) using a probe-tip sonicator, then centrifuged (13,000 ×
g, 10 min) and supernatant was stored at −80 °C until analysis. On the day of analysis total protein concentration was determined using a BCA assay. Total cell lysates (12 μg of protein) were solubilized in LDS (lithium dodecyl sulfate) buffer under reducing conditions. Proteins were separated by SDS-PAGE using a 12% Bis–Tris gel and transferred to a PVDF membrane. Following blocking with
blocking buffer (LI-COR Biosciences), membranes were incubated overnight with primary antibodies (alpha-synuclein: 1:1000,
Syn-1, BD Biosciences;
GAPDH: GAPDH, 1:10,000, Millipore). A near infrared fluorescent-labeled secondary antibody (1:5000;
IR800CW; LI-COR Biosciences) was used and quantification was done with an
Odyssey CLx infrared imaging system (LI-COR Biosciences) and
ImageJ/Fiji (NIH).
Schaser A.J., Stackhouse T.L., Weston L.J., Kerstein P.C., Osterberg V.R., López C.S., Dickson D.W., Luk K.C., Meshul C.K., Woltjer R.L, & Unni V.K. (2020). Trans-synaptic and retrograde axonal spread of Lewy pathology following pre-formed fibril injection in an in vivo A53T alpha-synuclein mouse model of synucleinopathy. Acta Neuropathologica Communications, 8, 150.
