Syn211

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The Syn211 is a laboratory instrument designed for sample preparation and analysis. It is a multi-purpose device that can be utilized for various applications within the scientific research and industrial settings. The Syn211 offers core functionalities to support essential laboratory workflows, however, a detailed description of its intended use is not available at this time.

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Lab products found in correlation

Versadoc xl imaging apparatus, by Bio-Rad (3 mentions) β actin, by Merck Group (3 mentions) Clone ac 15, by Merck Group (1 mentions) Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions) Lb509, by Fortrea (1 mentions) Secondary antibodies tagged with horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions) Human neurosin, by R&D Systems (1 mentions) Duolink assay kit, by Olink (1 mentions) Sc 14002, by Santa Cruz Biotechnology (1 mentions) Hoechst 33258, by Merck Group (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions) Chemidoc, by Bio-Rad (1 mentions) Supersignal west femto max sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Roti block, by Carl Roth (1 mentions) Ep1532y, by Abcam (1 mentions) Envisiontm system, by Agilent Technologies (1 mentions) Ep1536y, by Abcam (1 mentions) Cd235a, by BioLegend (1 mentions) Anti parvalbumin, by Merck Group (1 mentions) Anti neun, by Merck Group (1 mentions)

11 protocols using syn211

Immunodetection of α-Synuclein Variants

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For α-synuclein detection, mouse monoclonal antibody Syn-1 (BD Biosciences, San Diego, CA, USA) and rabbit polyclonal antibody C-20 (Santa Cruz Biotechnology, Dallas, TX, USA) were used. For specific detection of human α-synuclein, mouse monoclonal antibody Syn211 (Thermo Fisher Scientific, UK) was used, because Syn211 recognizes human α-synuclein, but not mouse α-synuclein [18] (link). Phosphorylated α-synuclein was detected by mouse monoclonal antibody pSyn#64 (Wako Pure Chemical Industries, Japan) or rabbit polyclonal antibody phospho-S129 (Abcam, UK). Glutamic acid decarboxylase (GAD) was detected by rabbit polyclonal antibodies purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-synapsin antibody was produced as previously described [19] . The following antibodies were purchased from these manufacturers: anti-synaptotagmin (Developmental Studies Hybridoma Bank, Iowa city, IA, USA), anti-vesicular glutamate transporter-1 (vGluT-1; Millipore, Billerica, MA, USA), anti-parvalbumin (Sigma-Aldrich), anti-somatostatin (Millipore), and anti-NeuN (Millipore).

Taguchi K., Watanabe Y., Tsujimura A., Tatebe H., Miyata S., Tokuda T., Mizuno T, & Tanaka M. (2014). Differential Expression of Alpha-Synuclein in Hippocampal Neurons. PLoS ONE, 9(2), e89327.

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Quantification of Phosphorylated α-Synuclein

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The western blot procedures were as previously employed.^{14 (link),15 (link)} Details of antibodies used in this study are available upon request. A previously characterized rabbit polyclonal antibody raised against p-α-syn (phosphorylated at serine 129) was used for immunodetection of p-α-syn.^{3 (link),14 (link),15 (link),27 (link)} Total α-synuclein was measured by western blot procedures using a non-phosphorylation-dependent antibody that detects an epitope between amino acid 121-125 of human α-synuclein (Syn 211, Life Technologies). The intensity of p-α-syn and total α-synuclein bands at 16 kD (monomeric) were quantified. Other proteins analyzed include Aβ peptide, phosphorylated tau, and synaptic proteins (SNAP-25 and synaptophysin). The level of β-actin in each sample (detected with Clone AC-15, Sigma, 1:10,000) was used for normalization purposes. The intensity of the immunoreactive bands for all antibodies were produced by reaction of blots with chemiluminescent substrate were measured using a direct imaging CCD camera system (Fluorochem Q, Protein Simple, San Jose, CA) and AlphaView Software. This system has a dynamic range of 4 logs to ensure linearity of dose-response. This was confirmed by using multiple exposures of each analysis.

Caviness J.N., Lue L.F., Hentz J.G., Schmitz C.T., Adler C.H., Shill H.A., Sabbagh M.N., Beach T.G, & Walker D.G. (2016). Cortical Phosphorylated α-Synuclein Levels Correlate with Brain Wave Spectra in Parkinson's Disease. Movement disorders : official journal of the Movement Disorder Society, 31(7), 1012-1019.

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Immunoblot Analysis of α-Synuclein in Transgenic Mice

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Hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [54 (link),55 (link)]. For immunoblot analysis, 20 μg of total protein per lane was loaded on 4-12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. To determine the effects of the immunotherapy in levels of α-syn, blotted samples from immunized α-syn tg mice were probed with antibodies against full length human α-syn (1:1000, SYN211, Life Technologies). Incubation with primary antibody was followed by species-appropriate incubation with secondary antibody tagged with horseradish peroxidase (1:5000, Santa Cruz Biotechnology), visualization with enhanced chemiluminescence, and analysis with a Versadoc XL imaging apparatus (BioRad). Analysis of β-actin (Sigma) levels was used as a loading control.
For studying which species the antibodies elicited by AFF 1 recognize, recombinant or 4-hydroxy-2-nonenal-treated α-syn [25 (link)] were loaded on 4-12% Bis-Tris SDS-PAGE gels and analyzed by immunoblot using AFF 1-elicited antibodies as primary antibody. The monoclonal antibody LB509 (Covance) served as positive control.

Mandler M., Valera E., Rockenstein E., Mante M., Weninger H., Patrick C., Adame A., Schmidhuber S., Santic R., Schneeberger A., Schmidt W., Mattner F, & Masliah E. (2015). Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy. Molecular Neurodegeneration, 10, 10.

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Immunotherapy Effects on α-Synuclein Levels

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Hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [72 (link),14 (link)]. For immunoblot analysis, 20 μg of total protein per lane was loaded on 4–12% Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. To determine the effects of the immunotherapy in levels of α-syn, blotted samples from immunized α-syn tg mice were probed with antibodies against full length human α-syn (1:1000, SYN211, Life Technologies). Additional analysis was performed with antibodies against β-syn (Abcam). Incubation with primary antibodies was followed by species-appropriate incubation with secondary antibodies tagged with horseradish peroxidase (1:5000, Santa Cruz Biotechnology), visualization with enhanced chemiluminescence, and analysis with a Versadoc XL imaging apparatus (BioRad). Analysis of β-actin (Sigma) levels was used as a loading control.
To determine human α-syn levels, an ELISA analysis was used (Life Technologies), performed following manufacturer’s instructions. This ELISA kit is designed to react with human α-syn specifically, without detectable cross-reactivity to mouse α-syn.

Mandler M., Valera E., Rockenstein E., Weninger H., Patrick C., Adame A., Santic R., Meindl S., Vigl B., Smrzka O., Schneeberger A., Mattner F, & Masliah E. (2014). Next-generation active immunization approach for synucleinopathies - implications for Parkinson’s Disease clinical trials. Acta neuropathologica, 127(6), 861-879.

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Immunoblot Analysis of α-Synuclein and Neurosin

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Hemibrains were homogenized and divided into cytosolic and membrane fractions as previously described [54 (link), 58 (link)]. For immunoblot analysis, 20 μg of total protein per lane was loaded on 4–12 % Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes. Membranes were probed with antibodies against full-length human α-syn (SYN211, Life Technologies), human neurosin (R&D Systems) or the epitope tag V5 (Life Technologies). Incubation with primary antibody was followed by species-appropriate incubation with secondary antibody tagged with horseradish peroxidase (Santa Cruz Biotechnology) and visualization with enhanced chemiluminescence.
Heparinized blood (plasma) (diluted 1:10 in PBS) and CSF (undiluted) samples were loaded on 4-12 % Bis-Tris SDS-PAGE gels and blotted onto polyvinylidene fluoride membranes and probed with antibodies against human neurosin (R&D Systems) as described above. Analysis of all immunoblot was performed with a Versadoc XL imaging apparatus (BioRad) using β-actin (Sigma) levels as a loading control.

Spencer B., Valera E., Rockenstein E., Trejo-Morales M., Adame A, & Masliah E. (2015). A brain-targeted, modified neurosin (kallikrein-6) reduces α-synuclein accumulation in a mouse model of multiple system atrophy. Molecular Neurodegeneration, 10, 48.

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In Situ PLA for Protein-Protein Interactions

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The in situ PLA allows the detection of protein-protein interactions in situ in intact tissues [7 (link),27 (link),30 (link),31 (link),32 (link),33 (link),48 (link)]. For the fluorescence human in situ PLA studies, we analyzed paraffin embedded brain sections from the caudate putamen of PD patients and age-matched controls by using the Duolink assay kit (O-Link Bioscience, Uppsala, Sweden) with a protocol adapted from the manufacturer’s instructions. Briefly, following deparaffinization and antigen retrieval with 10 mM sodium citrate buffer, sections were incubated in a blocking solution (provided by the kit) for 1 h at rt and then with the primary antibodies recognizing DAT (sc-14002, Santa Cruz Biotechnology), and α-synuclein (MA5-12272, Syn211, Thermo Fisher Scientific) at 1:100 dilution at 4 °C. On the following day, samples were washed and then incubated with a PLA probe solution for 1 h at rt. Sections were then washed and incubated with the ligation solution for 45 min at 37 °C, and then with the amplification solution at 37 °C for 100 min. Finally, cell nuclei were then counterstained with Hoechst 33258 (Sigma-Aldrich), and sections were mounted using the Vectashield mounting medium (Vector Laboratories).

Longhena F., Faustini G., Missale C., Pizzi M, & Bellucci A. (2018). Dopamine Transporter/α-Synuclein Complexes Are Altered in the Post Mortem Caudate Putamen of Parkinson’s Disease: An In Situ Proximity Ligation Assay Study. International Journal of Molecular Sciences, 19(6), 1611.

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Dot-Blot Analysis of Alpha-Synuclein Antibodies

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The indicated amounts of recombinant αSyn were spotted onto nitrocellulose membranes (Amersham Protan 0.2 µM, Chalfont St. Giles, Great Britain), dried for 15 min and blocked for 1 hour at 20 °C once in RotiBlock (Carl Roth, Karlsruhe, Germany). Each dot blot was incubated with either 0.2 µg/mL nAbs-αSyn, FT or anti-αSyn antibody (1:2000, Syn 211, Thermo Scientific, Rockford, USA) over night at 4 °C and washed 3 times with Tris buffered saline with 0.05% Tween-20 (TBS-T) for 30 min. As secondary antibody, horse reddish peroxidase (HRP) coupled to anti-human IgG antibodies (Peroxidase conjugated Affinity Pure Anti-Human IgG, Jackson Immunoresearch, Ely, United Kingdom) was used to detect the antibodies of human source (nAbs-αSyn and FT). Anti-mouse antibodies (Peroxidase conjugated Affinity Pure Goat anti-mouse IgG H + L, Jackson Immunoresearch, Ely, United Kingdom) were used to detect the anti-αSyn antibody. Blots were developed with Super Signal West Femto Max Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) on Bio-Rad ChemiDoc. (Bio-Rad, Munich, Germany). The experimental setup is schematically depicted in Figure 1a.

Braczynski A.K., Sevenich M., Gering I., Kupreichyk T., Agerschou E.D., Kronimus Y., Habib P., Stoldt M., Willbold D., Schulz J.B., Bach J.P., Falkenburger B.H, & Hoyer W. (2022). Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo. Biomolecules, 12(3), 469.

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Histological Analysis of Striatum and Midbrain

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Immediately after completion of the behavioral tests, i.e. at 12 weeks post-surgery, mice were anesthetized with sodium pentobarbital (100 mg/kg body weight, i.p.) and perfused with 0.9% NaCl followed by ice cold 4% paraformaldehyde (PFA). Brains were post-fixed for 24 h in 4% PFA, then cryoprotected in 20% sucrose in 0.1M PBS. Brains were frozen in isopentane and stored at -80 °C. 50 μm free-floating coronal sections of the entire striatum and midbrain were collected for histological analysis. Immunohistochemistry was performed for each animal on striatum (from 1 to 3 sections) and on every fourth midbrain sections spanning the entire rostro-caudal SNpc. Selected sections were incubated all together overnight with primary antibodies for the following antigens: TH (1:5.000 clone EP1532Y, ab137869 Abcam), α-synuclein (1:1.000 clone Syn211, Thermo Scientific, MS1572), or phosphorylated Ser129 α-synuclein (1:5.000, EP1536Y, ab51253, Abcam). Immunoreactions were revealed by an anti–species dependent peroxidase EnVisionTM system (DAKO) followed by DAB visualization. Sections were mounted on gelatinized slides, counterstained with 0.1% cresyl violet solution if needed, dehydrated, and cover-slipped.

Sun X., Yu X., Zhang L., Zhao W., Wang M., Zhang Y., Li X., Gao R., Breger L.S., Dovero S., Porras G., Fernagut P.O., Dehay B., Bezard E, & Qin C. (2021). Comparison of the expression and toxicity of AAV2/9 carrying the human A53T α-synuclein gene in presence or absence of WPRE. Heliyon, 7(2), e06302.

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Quantifying Extracellular Vesicles in Plasma

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Stored plasma samples were thawed and centrifuged at 13,000 x g for 5 minutes to remove aggregates. Plasmas were diluted 1/10^th in PBS, and 10 microliters of diluted plasma were incubated with fluorescent-labeled primary antibodies against total α-synuclein (syn211, Thermo Fisher) and CD235a (clone HI264, Biolegend). EV populations were quantified from platelet-free plasma by nanoscale-flow cytometry using the Apogee A60-Micro Plus (Apogee Flow Systems Inc.). Before sample analysis, A60-Micro Plus sensitivity for detecting EVs was evaluated using a reference bead mix, as previously described [11 (link)] (S1 Fig). Sample flow rate was set at 1.5μL/min for all measurements, and acquisition time held constant for all samples at 60 seconds. Event rate was kept below 5,000 events per second to avoid swarm effect. Antibody-matched isotypes in the same condition were used to subtract unspecific binding. Data were analyzed using FlowJo v10.5 software. A one-way ANOVA test and a nonparametric t test identified differences between the control and patient groups in terms of clinical variables.

Lucien F., Benarroch E.E., Mullan A., Ali F., Boeve B.F., Mielke M.M., Petersen R.C., Kim Y., Stang C., Camerucci E., Ross O.A., Wszolek Z.K., Knopman D., Bower J., Singer W, & Savica R. (2022). Poly (ADP-Ribose) and α–synuclein extracellular vesicles in patients with Parkinson disease: A possible biomarker of disease severity. PLoS ONE, 17(4), e0264446.

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Immunohistochemical Detection of Neurological Markers

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IHC Detection System Kit (ZSGB‐BIO, PV‐6001/PV‐6002) was used. The sections were incubated with primary antibodies against TH (1:2000, Abcam, ab117112), pS129 (1:1000, Biolegend, 825701), IBA1 (1:500, Wako, 019–19741), GFAP (1:500, Thermo Fisher Scientific, PA5‐16291), Syn211 (1:1000, Thermo Fisher Scientific, MA5‐12272), or K80Hcy antibody (1:500, Abmart) at 4°C for overnight. The signal was developed using DAB. The levels of immunoreactivity were determined by optical density analysis using Image J, plus the IHC Profiler plugin.

Zhou L., Guo T., Meng L., Zhang X., Tian Y., Dai L., Niu X., Li Y., Liu C., Chen G., Liu C., Ke W., Zhang Z., Bao A, & Zhang Z. (2022). N‐homocysteinylation of α‐synuclein promotes its aggregation and neurotoxicity. Aging Cell, 22(3), e13745.

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