Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11]

Mouse Anti-Mouse Alpha Synuclein Aggregate Antibody Monoclonal IgG1

Catalog No. SMC-617

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Species Reactivity Hu, Ms
Applications WB IHC ICC/IF FCM IP
SKU: SMC-617 Categories: ,

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SMC-617_Alpha-Synuclein-Aggregate-Specific_Antibody_2F11_Immuno-Gold-Labelling_1.png
Mouse Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11] used in Binding Analysis () on (SMC-617)Mouse Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11] used in Immunocytochemistry/Immunofluorescence (ICC/IF) on iPSC (SMC-617)Mouse Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11] used in Immunocytochemistry/Immunofluorescence (ICC/IF) on Human SH-SY5Y (SMC-617)Mouse Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11] used in Immunohistochemistry (IHC) on Mouse gut (SMC-617)Mouse Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11] used in Western Blot (WB) on Mouse brain lysate (SMC-617)Mouse Anti-Alpha Synuclein Antibody (Aggregate-Specific) [2F11] used in Western Blot (WB) on Human HeLa (SMC-617)
Product Name Alpha Synuclein Antibody (Aggregate-Specific)
Description

Mouse Anti-Mouse Alpha Synuclein Aggregate Antibody Monoclonal IgG1
Species Reactivity Human, Mouse
Applications WB, DB, IP, IHC, ICC-IF, ImmunoGold labelling
Antibody Dilution WB (1:1000). Application specific; optimal dilutions for assays should be determined by the user.
Host Species Mouse
Immunogen Species Mouse
Immunogen Mouse alpha synuclein fibrils
Concentration 1mg/mL
Conjugates APC, ATTO 390, ATTO 488, ATTO 594, Biotin, FITC, HRP, PerCP, RPE, Unconjugated
Dylight 488
Overview:

  • High fluorescence yield
  • High photostability
  • Less pH-sensitive
  • Excellent batch-to-batch reproducibility
  • Stringently QC tested
  • Molecular weight: 1011 g/mol

Dylight 488 Datasheet

 Dylight 488 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 493 nm

λem = 518 nm

εmax = 7.0×104

Laser = 488 nm

 

APC/Cy7
Overview:

  • High quantum yield
  • Excellent batch-to-batch reproducibility
  • Stringently QC tested

APC-Cy7 Datasheet

 

 ACP-Cy7 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 652 nm

λem = 790 nm

Laser = 594 or 633 nm

 

 

  Dylight 350
Overview:

  • High fluorescence intensity
  • High photostability
  • Less pH-sensitive
  • Excellent solubility in water
  • Stringently QC tested
  • Excellent batch-to-batch reproducibility
  • Molecular weight: 874 g/mol

Dylight 350 Datasheet

 Dylight 350 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 353 nm

λem = 432 nm

εmax = 1.5×104

 

 

  Dylight 405
Overview:

  • High fluorescence intensity
  • High photostability
  • Less pH-sensitive
  • Excellent batch-to-batch reproducibility
  • Stringently QC tested
  • Molecular weight: 793 g/mol

Dylight 405 Datasheet

 Dylight 405 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 400 nm

λem = 420 nm

εmax = 3.0×104

Laser = 405 nm

 

Dylight 594
Overview:

  • High fluorescence yield
  • High photostability
  • Less pH-sensitive
  • Excellent batch-to-batch reproducibility
  • Stringently QC tested
  • Molecular weight: 1078 g/mol

Dylight 594 Datasheet

 Dylight 594 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 593 nm

λem = 618 nm

εmax = 8.0×104

Laser = 526 nm

 

 Dylight 633
Overview:

  • High fluorescence yield
  • High photostability
  • Less pH-sensitive
  • Excellent batch-to-batch reproducibility
  • Stringently QC tested
  • Molecular weight: 1066 g/mol

Dylight 633 Datasheet

 Dylight 633 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 638 nm

λem = 658 nm

εmax = 1.7×105

Laser = 633 nm

 

 PerCP 
Overview:

  • Peridinin-Chlorophyll-Protein Complex
  • Small phycobiliprotein
  • Isolated from red algae
  • Large stokes shift (195 nm)
  • Molecular Weight: 35 kDa
  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

PerCP Datasheet

  PerCP Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 482 nm

λem = 677 nm

εmax = 1.96 x 106

Laser = 488 nm

 

 PE/ATTO 594
PE/ATTO 594 is a tandem conjugate, where PE is excited at 535 nm and transfers energy to ATTO 594 via FRET (fluorescence resonance energy transfer), which emits at 627 nm.
Overview:

  • High fluorescence yield
  • High photostability
  • Very hydrophilic
  • Excellent solubility in water
  • Very little aggregation

PE/ATTO 594 Datasheet

 PE-ATTO 594 Fluorophore Conjugate Excitation and Emission Spectra Optical Properties:

λex = 535 nm

λem = 627 nm

Laser = 488 to 561 nm

 

  FITC (Fluorescein)
Overview:

  • Excellent fluorescence quantum yield
  • High rate of photobleaching
  • Good solubility in water
  • Broad emission spectrum
  • pH dependent spectra
  • Molecular formula: C20H12O5
  • Molar mass: 332.3 g/mol
  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

FITC-Fluorescent-conjugate

 FITC Fluorescein Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 494 nm

λem = 520 nm

εmax = 7.3×104

Φf = 0.92

τfl = 5.0 ns

Brightness = 67.2

Laser = 488 nm

Filter set = FITC

 

 ATTO 700
Overview:

  • High fluorescence yield
  • Excellent thermal and photostability
  • Quenched by electron donors
  • Very hydrophilic
  • Good solubility in polar solvents
  • Zwitterionic dye
  • Molar Mass: 575 g/mol

ATTO 700 Datasheet

  ATTO 700 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 700 nm

λem = 719 nm

εmax = 1.25×105

Φf = 0.25

τfl = 1.6 ns

Brightness = 31.3

Laser = 676 nm

Filter set = Cy®5.5

 

 ATTO 680
Overview:

  • High fluorescence yield
  • Excellent thermal and photostability
  • Quenched by electron donors
  • Very hydrophilic
  • Good solubility in polar solvents
  • Zwitterionic dye
  • Molar Mass: 631 g/mol

ATTO 680 Datasheet

  ATTO 680 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 680 nm

λem = 700 nm

εmax = 1.25×105

Φf = 0.30

τfl = 1.7 ns

Brightness = 37.5

Laser = 633 – 676 nm

Filter set = Cy®5.5

 

 ATTO 655
Overview:

  • High fluorescence yield
  • High thermal and photostability
  • Excellent ozone resistance
  • Quenched by electron donors
  • Very hydrophilic
  • Good solubility in polar solvents
  • Zwitterionic dye
  • Molar Mass: 634 g/mol

ATTO 655 Datasheet

 ATTO 655 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 663 nm

λem = 684 nm

εmax = 1.25×105

Φf = 0.30

τfl = 1.8 ns

Brightness = 37.5

Laser = 633 – 647 nm

Filter set = Cy®5

 

 ATTO 633
Overview:

  • High fluorescence yield
  • High thermal and photostability
  • Moderately hydrophilic
  • Good solubility in polar solvents
  • Stable at pH 4 – 11
  • Cationic dye, perchlorate salt
  • Molar Mass: 652.2 g/mol

ATTO 633 Datasheet

 ATTO 633 Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 629 nm

λem = 657 nm

εmax = 1.3×105

Φf = 0.64

τfl = 3.2 ns

Brightness = 83.2

Laser = 633 nm

Filter set = Cy®5

 

 ATTO 594
Overview:

  • High fluorescence yield
  • High photostability
  • Very hydrophilic
  • Excellent solubility in water
  • Very little aggregation
  • New dye with net charge of -1
  • Molar Mass: 1137 g/mol
  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

ATTO 594 Datasheet

  ATTO 594 Fluorophore Excitation and Emission Spectrum Optical Properties:

λex = 601 nm

λem = 627 nm

εmax = 1.2×105

Φf = 0.85

τfl = 3.5 ns

Brightness = 102

Laser = 594 nm

Filter set = Texas Red®

 

 ATTO 565
Overview:

  • High fluorescence yield
  • High thermal and photostability
  • Good solubility in polar solvents
  • Excellent solubility in water
  • Very little aggregation
  • Rhodamine dye derivative
  • Molar Mass: 611 g/mol

ATTO 565 Datasheet

  ATTO 565 Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 563 nm

λem = 592 nm

εmax = 1.2×105

Φf = 0.9

τfl = 3.4 n

Brightness = 10

Laser = 532 nm

Filter set = TRITC

 

  ATTO 488
Overview:

  • High fluorescence yield
  • High photostability
  • Very hydrophilic
  • Excellent solubility in water
  • Very little aggregation
  • New dye with net charge of -1
  • Molar Mass: 804 g/mol
  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications. 

ATTO 488 Datasheet

   ATTO 488 Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 501 nm

λem = 523 nm

εmax = 9.0×104

Φf = 0.80

τfl = 4.1 ns

Brightness = 72

Laser = 488 nm

Filter set = FITC

 

  ATTO 390
Overview:

  • High fluorescence yield
  • Large Stokes-shift (89 nm)
  • Good photostability
  • Moderately hydrophilic
  • Good solubility in polar solvents
  • Coumarin derivate, uncharged
  • Low molar mass: 343.42 g/mol
  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications. 

ATTO 390 Datasheet

 ATTO 390 Fluorescent Dye Excitation and Emission Spectra Optical Properties:

λex = 390 nm

λem = 479 nm

εmax = 2.4×104

Φf = 0.90

τfl = 5.0 ns

Brightness = 21.6

Laser = 365 or 405 nm

 

APC (Allophycocyanin)
Overview:

  • High quantum yield
  • Large phycobiliprotein
  • 6 chromophores per molecule
  • Isolated from red algae
  • Molecular Weight: 105 kDa
  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

APC Datasheet

  APC Fluorophore Absorption and Emission Spectrum Optical Properties:

λex = 650 nm

λem = 660 nm

εmax = 7.0×105

Φf = 0.68

Brightness = 476

Laser = 594 or 633 nm

Filter set = Cy®5

 

Streptavidin

Properties:

  • Homo-tetrameric protein purified from Streptomyces avidinii which binds four biotin molecules with extremely high affinity
  • Molecular weight: 53 kDa
  • Formula: C10H16N2O3S
  • Applications: Western blot, immunohistochemistry, and ELISA

Streptavidin Datasheet

BiotinBiotin Conjugate Structure

Properties:

  • Binds tetrameric avidin proteins including Streptavidin and neuravidin with very high affinity
  • Molar mass: 244.31 g/mol
  • Formula: C10H16N2O3S
  • Applications: Western blot, immunohistochemistry, and ELISA*

*The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

Biotin Datasheet

HRP (Horseradish peroxidase)

Properties:

  • Enzymatic activity is used to amplify weak signals and increase visibility of a target
  • Readily combines with hydrogen peroxide (H2O2) to form HRP-H2O2 complex which can oxidize various hydrogen donors
  • Catalyzes the conversion of:
    • Chromogenic substrates (e.g. TMB, DAB, ABTS) into coloured products
    • Chemiluminescent substrates (e.g. luminol and isoluminol) into light emitting products via enhanced chemiluminescence (ECL)
    • Fluorogenic substrates (e.g. tyramine, homovanillic acid, and 4-hydroxyphenyl acetic acid) into fluorescent products
  • High turnover rate enables rapid generation of a strong signal
  • 44 kDa glycoprotein
  • Extinction coefficient: 100 (403 nm)
  • Applications: Western blot, immunohistochemistry, and ELISA*
    • *The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

HRP Datasheet

AP (Alkaline Phosphatase)

Properties:

  • Broad enzymatic activity for phosphate esters of alcohols, amines, pyrophosphate, and phenols
  • Commonly used to dephosphorylate the 5’-termini of DNA and RNA to prevent self-ligation
  • Catalyzes the conversion of:
    • Chromogenic substrates (e.g. pNPP, naphthol AS-TR phosphate, BCIP) into coloured products
    • Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate) into fluorescent products
  • Molecular weight: 140 kDa
  • Applications: Western blot, immunohistochemistry, and ELISA

AP Datasheet

  R-PE (R-Phycoerythrin)
Overview:

  • Broad excitation spectrum
  • High quantum yield
  • Photostable
  • Member of the phycobiliprotein family
  • Isolated from red algae
  • Excellent solubility in water
  • Molecular Weight: 250 kDa

 

  • Applications: The listed applications provide a general overview of potential uses for conjugated antibodies. However, they do not guarantee that every antibody-conjugate combination has been tested or validated for these specific applications.

R-PE Datasheet

  R-PE Fluorophore Excitation and Emission Spectra Optical Properties:

λex = 565 nm

λem = 575 nm

εmax = 2.0×106

Φf = 0.84

Brightness = 1.68 x 103

Laser = 488 to 561 nm

Filter set = TRITC

 
Field of Use Not for use in humans. Not for use in diagnostics or therapeutics. For in vitro research use only.

Properties

Storage Buffer PBS pH 7.4, 50% glycerol, 0.09% sodium azide. *Storage buffer may change when conjugated
Storage Temperature -20ºC, Conjugated antibodies should be stored according to the product label
Shipping Temperature Blue Ice or 4ºC
Purification Protein G Purified
Clonality Monoclonal
Clone Number 2F11
Isotype IgG1
Specificity This antibody binds a 3-dimensional fibrillar epitope of alpha synuclein (including AA101-120, AA61-80), present in MSA (Multiple System Atrophy) aggregates in human brain samples. It does not appear to detect PD (Parkinson's disease) or PDLB (Parkinson's disease with Lewy Bodies) aggregate structures in human brain samples. It can, however, bind recombinant human PFFs and mouse PFFs.
Cite This Product Mouse Anti-Mouse Alpha Synuclein Aggregate Antibody Monoclonal (StressMarq Biosciences, Victoria BC, Cat# SMC-617)
Certificate of Analysis A 1:1000 dilution of SMC-617 was sufficient for detection of Alpha Synuclein aggregates in 15 µg of human brain cell lysate by ECL immunoblot analysis using goat anti-mouse IgG:HRP as the secondary antibody.

Biological Description

Alternative Names Alpha Synuclein, α-Synuclein, SNCA, Snca, SYN, alphaSYN, NACP, Non-A beta component of AD amyloid, Non-A4 component of amyloid precursor, Parkinson disease familial 1, PARK1, PARK 1, PARK4, PARK 4, Parkinson disease (autosomal dominant, Lewy body) 4, Lewy body 4, Synuclein-alpha, PD1
Research Areas Alzheimer's Disease, Neurodegeneration, Neuroscience, Synuclein, Tangles & Tau, Multiple System Atrophy
Cellular Localization Cell Junction, Cytoplasm, Cytosol, Membrane, Nucleus, Synapse
Accession Number NP_001035916.1
Gene ID 20617
Swiss Prot O55042
Scientific Background Alpha-synuclein (SNCA) is a presynaptic neuronal protein abundantly expressed in the brain, particularly within the olfactory bulb, hippocampus, striatum, and thalamus (1,2). Localized to both synaptic terminals and mitochondria, alpha-synuclein plays a critical role in synaptic vesicle regulation and cytoskeletal dynamics through its interaction with tubulin (3), suggesting a potential function as a microtubule-associated protein. It is also essential for cognitive development, with SNCA inactivation linked to deficits in spatial learning and working memory (4). Pathologically, alpha-synuclein aggregates are a defining feature of several neurodegenerative diseases. These fibrillar inclusions constitute the major non-Aβ component of amyloid plaques in Alzheimer’s disease and are the principal constituents of Lewy bodies in Parkinson’s disease (PD). In PD, progressive accumulation of alpha-synuclein and ubiquitin-positive inclusions in dopaminergic neurons is closely associated with motor and cognitive decline (5,6). Alpha-synuclein aggregation is not merely a byproduct of disease but a driver of neurotoxicity, mitochondrial dysfunction, and synaptic failure. Its central role in disease pathogenesis has made it a high-priority target for biomarker development and therapeutic intervention. Covered by US patent family US11,098,108B2 (7).
References 1. “Genetics Home Reference: SNCA”. US National Library of Medicine. (2013).
2. Zhang L., et al. (2008) Brain Res. 1244: 40-52.
3. Alim M.A., et al. (2002) J Biol Chem. 277(3): 2112-2117.
4. Kokhan V.S., Afanasyeva M.A., Van'kin G. (2012) Behav. Brain. Res. 231(1): 226-230.
5. Spillantini M.G., et al. (1997) Nature. 388(6645): 839-840.
6. Mezey E., et al. (1998) Nat Med. 4(7): 755-757.
7. Louwrier, A. US Patent family US11,098,108 B2

Product Images

<p>Immuno-gold electron micrograph of sonicated α-Syn Fibrils (SPR-322) stained with 2F11 antibody (1:400 dilution) and visualized using goat anti-mouse IgG conjugated with 18 nm gold particles. The sample was then placed on a transmission electron microscopy metallic grid and stained with uranyl acetate.</p>

Immuno-gold electron micrograph of sonicated α-Syn Fibrils (SPR-322) stained with 2F11 antibody (1:400 dilution) and visualized using goat anti-mouse IgG conjugated with 18 nm gold particles. The sample was then placed on a transmission electron microscopy metallic grid and stained with uranyl acetate.

<p>Western blot on 15ug mouse brain lysate from Fmr1 KO mice (FXS mice) with C57Bl/6J genetic background. Block: 5% Skim Milk in 1X TBST. Primary Antibody: Mouse Anti-Alpha Synuclein Monoclonal Antibody clone 2F11 at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse HRP:IgG at 1:3000 for 1 hour at RT. Color Development: ECL solution (Super Signal West Pico) for 5 min in RT. Band clearly identifies large molecular weight oligomeric band (monomeric band predicted at 14kD).</p>

Western blot on 15ug mouse brain lysate from Fmr1 KO mice (FXS mice) with C57Bl/6J genetic background. Block: 5% Skim Milk in 1X TBST. Primary Antibody: Mouse Anti-Alpha Synuclein Monoclonal Antibody clone 2F11 at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse HRP:IgG at 1:3000 for 1 hour at RT. Color Development: ECL solution (Super Signal West Pico) for 5 min in RT. Band clearly identifies large molecular weight oligomeric band (monomeric band predicted at 14kD).

<p>Western blot on 15ug Hela lysate. Block: 5% Skim Milk in 1X TBST. Primary Antibody: Mouse Anti-Alpha Synuclein Monoclonal Antibody clone 2F11 at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse HRP:IgG at 1:3000 for 1 hour at RT. Color Development: ECL solution (Super Signal West Pico) for 5 min in RT. Band clearly identifies large molecular weight oligomeric band (monomeric band predicted at 14kD).</p>

Western blot on 15ug Hela lysate. Block: 5% Skim Milk in 1X TBST. Primary Antibody: Mouse Anti-Alpha Synuclein Monoclonal Antibody clone 2F11 at 1:1000 for 2 hours at RT. Secondary Antibody: Goat Anti-Mouse HRP:IgG at 1:3000 for 1 hour at RT. Color Development: ECL solution (Super Signal West Pico) for 5 min in RT. Band clearly identifies large molecular weight oligomeric band (monomeric band predicted at 14kD).

<p>SPR binding analysis of 2F11 antibody and human alpha synuclein type 1 fibrils (SPR-322), type 2 (SPR-317) fibrils and monomeric protein (SPR-321). Buffer (top left), alpha synuclein monomers (top right), alpha synuclein fibrils type1 (bottom left), and alpha synuclein fibrils type 2 (bottom right). Data: KD[nM] for SPR-322 = 42.5, SPR-317 = 365.3; 2F11 binds SPR-322 with 8.6 x greater affinity than SPR-317. 2F11 does not bind monomeric alpha synuclein (SPR-321).</p>

SPR binding analysis of 2F11 antibody and human alpha synuclein type 1 fibrils (SPR-322), type 2 (SPR-317) fibrils and monomeric protein (SPR-321). Buffer (top left), alpha synuclein monomers (top right), alpha synuclein fibrils type1 (bottom left), and alpha synuclein fibrils type 2 (bottom right). Data: KD[nM] for SPR-322 = 42.5, SPR-317 = 365.3; 2F11 binds SPR-322 with 8.6 x greater affinity than SPR-317. 2F11 does not bind monomeric alpha synuclein (SPR-321).

<p>8 Day differentiated iPSCs with added alpha synuclein fibrils (SPR-322), conjugated to Alexa Fluor 555 with or without 1h prior 2F11 addition. SPR-322 Alexa Fluor 555 added at 5µg/well for 48h, 2F11 (50ug/well added 1h before fibrils where applicable). Cells were subsequently washed 48h after addition and imaged a following 24h later. Imaging and channels: 2F11 antibody: 647nm (using an anti-mouse-647 conjugate); Fibrils: 555nm, phosph-ser129 (SMC-600) 488nm, and Hoechst stain for DNA. Top two images: no fibrils, no 2F11 added; fibrils added, no 2F11 antibody. Fibrils are clearly visible in the cells as is enhanced phosph-ser129 signal. Bottom two images: no fibrils, 2F11 antibody added; fibrils and 2F11 antibody added. The image clearly shows 2F11 antibody addition stops cell entry of the 555-labelled fibrils.</p>

8 Day differentiated iPSCs with added alpha synuclein fibrils (SPR-322), conjugated to Alexa Fluor 555 with or without 1h prior 2F11 addition. SPR-322 Alexa Fluor 555 added at 5µg/well for 48h, 2F11 (50ug/well added 1h before fibrils where applicable). Cells were subsequently washed 48h after addition and imaged a following 24h later. Imaging and channels: 2F11 antibody: 647nm (using an anti-mouse-647 conjugate); Fibrils: 555nm, phosph-ser129 (SMC-600) 488nm, and Hoechst stain for DNA. Top two images: no fibrils, no 2F11 added; fibrils added, no 2F11 antibody. Fibrils are clearly visible in the cells as is enhanced phosph-ser129 signal. Bottom two images: no fibrils, 2F11 antibody added; fibrils and 2F11 antibody added. The image clearly shows 2F11 antibody addition stops cell entry of the 555-labelled fibrils.

<p>Mouse gut samples stained with 2F11 at 1:1000 dilution with overnight incubation at 4°C in PBS with 0.1% BSA, Secondary biotinylated anti-mouse antibody for 1 hour at RT also in 0.1% BSA PBS. VECTASTAIN ABC reagent kit was added after secondary incubation. Peroxidase HRP substrate was left on the gut tissue for 6 minutes. H&E staining of all tissue was performed at 1:1 with dH20, rinsed, then stained with blueing solution prior to dehydration. Imaging at 40 x magnification. Mouse tissue used from a mouse Parkinson’s disease (PD) seeding model based on C57BLJ/6 mice with brain injections of Human PD patient alpha-synuclein fractions. Mouse gut sections were used at a thickness of 3µm. Top panel shows (in biological order of mouse gut from ileum to descending colon) control secondary staining. Bottom panel shows gut samples from mice injected with human PD patient brain-derived alpha synuclein, also in biological order (ileum to descending colon). There is clear aggregate staining visible in the lower panel.</p>

Mouse gut samples stained with 2F11 at 1:1000 dilution with overnight incubation at 4°C in PBS with 0.1% BSA, Secondary biotinylated anti-mouse antibody for 1 hour at RT also in 0.1% BSA PBS. VECTASTAIN ABC reagent kit was added after secondary incubation. Peroxidase HRP substrate was left on the gut tissue for 6 minutes. H&E staining of all tissue was performed at 1:1 with dH20, rinsed, then stained with blueing solution prior to dehydration. Imaging at 40 x magnification. Mouse tissue used from a mouse Parkinson’s disease (PD) seeding model based on C57BLJ/6 mice with brain injections of Human PD patient alpha-synuclein fractions. Mouse gut sections were used at a thickness of 3µm. Top panel shows (in biological order of mouse gut from ileum to descending colon) control secondary staining. Bottom panel shows gut samples from mice injected with human PD patient brain-derived alpha synuclein, also in biological order (ileum to descending colon). There is clear aggregate staining visible in the lower panel.

<p>8 Day differentiated SH-SY5Y cells with added alpha synuclein monomers (SPR-321; 5µg) or fibrils (SPR-322; 5µg) at time zero and 3h time points. Staining and channels phospho-ser129 (ab51253) and secondary DaRb-647; 2F11 (1:1000 dilution, DaMs-555); Actin stain (Phalloidin dye: 488nm); DNA (Hoechst dye, 405nm). Top two images: T=0, T=3h, no signal change from 2F11 showing that alpha synuclein monomers do not bind the antibody. Bottom two images: T=0, T=3h. 2F11 channel clearly shows a large increase in fibril signal entering the cells by 3h, with a concomitant increase in phspho-Ser129 signal.</p>

8 Day differentiated SH-SY5Y cells with added alpha synuclein monomers (SPR-321; 5µg) or fibrils (SPR-322; 5µg) at time zero and 3h time points. Staining and channels phospho-ser129 (ab51253) and secondary DaRb-647; 2F11 (1:1000 dilution, DaMs-555); Actin stain (Phalloidin dye: 488nm); DNA (Hoechst dye, 405nm). Top two images: T=0, T=3h, no signal change from 2F11 showing that alpha synuclein monomers do not bind the antibody. Bottom two images: T=0, T=3h. 2F11 channel clearly shows a large increase in fibril signal entering the cells by 3h, with a concomitant increase in phspho-Ser129 signal.

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