Primary rat hippocampal neurons show lewy body inclusion formation when treated with Type 1 Alpha Synuclein Protein Pre-formed Fibrils (SPR-322) at 4 µg/ml (D-F), but not when treated with Type 2 Alpha Synuclein Protein Pre-formed Fibrils (SPR-317) at 4 µg/ml (A-C). Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Fixation: 4% formaldehyde from PFA. Primary Antibody: Mouse anti-pSer129 Antibody at 1:1000 24 hours at 4°C. 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 inclusions. Magnification: 20x.

ATTO633 fluorescently-labelled alpha synuclein PFFs (SPR-322) were taken up, transported into the soma, and induced alpha synuclein aggregation in mouse neurocortical primary cells. (A) Neurites filled with fluorescently-labelled alpha synuclein seeds in a microfluidic co-culture system after 24 hours. (B) Alpha synuclein seeds within the soma and neurites of mouse neurocortical primary cells after 24 hours. Experiment and imaging courtesy of Cellectricon.

Confocal imaging shows NeuN+ (mature) primary cortical neurons filled with ATTO633 fluorescently-labelled alpha synuclein PFFs (SPR-322). ATTO-633 alpha synuclein PFFs seed endogenous alpha synculein phosphorylation after 7 days. Experiment and imaging courtesy of Cellectricon.

Immunohistochemistry analysis of rat brain injected with Type 1 human alpha synuclein PFFs (SPR-322). Species: Female Sprague-Dawley Rat. Rat was injected with 16µg Type 1 human alpha synuclein PFFs (SPR-322) in each of 2 injection sites: AP+1.6, ML+2.4, DV-4.2 from skull; and AP-1.4, ML+0.2, DV-2.8 from skull. 30-days post-injection. Fixation: Saline perfusion followed by 4% PFA fixation for 48 hrs. Primary antibody: rabbit monoclonal anti-pSer129 alpha synuclein. Secondary Antibody: Biotin-SP Donkey Anti-Rabbit IgG (H+L) at 1:500 for 2 hours in cold room with shaking. ABC signal amplification, DAB staining. Magnification: 20X. Alpha synuclein pathology is seen in the striatum close to an injection site.