Recombinant Human Alpha-synuclein protein aggregate (Active)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

Immunohistochemical analysis of primary rat hippocampal neurons showing lewy body inclusion formation when treated with active Alpha Synuclein Protein Aggregate (ab218819) at 4 μg/ml (D-F), but not when treated with control Alpha Synuclein Protein Aggregate (ab218817) 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 hour at RT. Counterstain : Hoechst (blue) nuclear stain at 1/4000 for 1 hour at RT. Localization : Lewy body incluscions. Magnification : 20x.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

ab218819 was shown to be taken up by SH-SY5Y cells and transmitted to neuronal iPSCs within 14 days. Blue : Hoechst/DNA; Green : SHSY5Y-GFP; Red : Alpha-synuclein protein aggregate (ab218819); Purple : Tubulin.

• EM

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Electron Microscopy - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

TEM of active human alpha synuclein preformed fibrils (ab218819) (top) and control (inactive) human alpha synuclein preformed fibrils (ab218817) (bottom). Fibrils were sonicated and treated with uranyl acetate. The active fibrils are shorter than the inactive fibrils.

• FuncS

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Functional Studies - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

ab218819 seeds the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers.

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 μM of ab218819 is combined with 100 μM of alpha synuclein monomer, as compared to ab218819 alone and alpha synuclein monomer alone.

Thioflavin T ex = 450 nm, em = 485 nm.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

Immunohistochemistry analysis of rat brain injected with active human alpha synuclein PFFs (ab218819). Species : Female Sprague-Dawley Rat. Rat was injected with 2μL active human alpha synuclein PFFs (ab218819) 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. 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. Alpha synuclein pathology is seen in the striatum close to an injection site.

• EM

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Electron Microscopy - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

TEM of Type 1 Alpha Synuclein Pre-formed Fibrils (PFFs) (ab218819)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

Immunocytochemistry/Immunofluorescence analysis of human iPSC-derived neurons treated with alpha synuclein pre-formed fibrils (ab218819). Primary Antibody : Mouse Anti-Alpha Synuclein (pSer129) Monoclonal Antibody at 1 : 1000 for O/N at 4°C. Secondary Antibody : Anti-Rabbit : A488 at 1 : 1000 for 1 hour at RT. Magnification : 40X. Nuclear stain : Hoechst- 20 min, RT (blue). Actin stain : Phalloidin-647- 20 min, RT (magenta). 4K cells per well. Negative control; no fibrils added to well. B) 7 days after addition of active recombinant human pre-formed fibrils (Type 1). Fibrils were sonicated before use and applied 2.5 ug per well

• SDS-PAGE

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SDS-PAGE - Recombinant Human Alpha-synuclein protein aggregate (Active) (AB218819)

SDS-PAGE analysis of ab218819.