Primary rat hippocampal neurons show lewy body inclusion formation when treated with active Alpha Synuclein Preformed Fibrils (GTX17669-pro) at 4 μg/ml (D-F), but not when treated with control Alpha Synuclein Preformed Fibrils (GTX17667-pro) at 4 μg/ml (A-C).
Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Fixation: 4% formaldehyde made from PFA. Primary Antibody: Mouse anti-pSer129 Antibody at 1:1000 24 hours at 4ºC. Secondary Antibody: FITC Goat Anti-Mouse (green) at 1:700 for 1 hours at RT. Counterstain: Hoechst (blue) nuclear stain at 1:4000 for 1 hour at RT. Localization: Lewy body incluscions. Magnification: 20x.

Active alpha synuclein preformed fibrils (GTX17669-pro) seed the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers (GTX17668-pro). Thioflavin T is a fluorescent dye that binds to beta sheet-rich structures, such as those in alpha synuclein fibrils. Upon binding, the emission spectrum of the dye experiences a red-shift and increased fluorescence intensity. Thioflavin T emission curves show increased fluorescence (correlated to alpha synuclein protein aggregation) over time when 10 nM of active alpha synuclein preformed fibrils (GTX17669-pro) is combined with 100 μM of alpha synuclein monomer (GTX17668-pro), as compared to when 10 nM of control alpha synuclein preformed fibrils (GTX17667-pro) is combined with 100 μM of alpha synuclein monomer (GTX17668-pro) or 100 μM of alpha Synuclein monomer (GTX17666-pro). Thioflavin T ex = 450 nm, em = 485 nm.

SDS-PAGE of ~14 kDa Human alpha Synuclein protein (fibrils) (GTX17667-pro).
Lane 1: Molecular Weight Ladder (MW). Lane 2: Human alpha Synuclein protein (fibrils) (GTX17667-pro).