The ventral midbrain tissues were dissected and prepared in lysis buffer that consist of 10 mM Tris-HCL, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-β-glycerophosphate, Phosphate inhibitor mixture I and II (Sigma-Aldrich, St. Louis, MO, USA), and complete protease inhibitor mixture (Roche) at pH 7.4. Then the tissues were homogenized using a Diax 900 homogenizer (Sigma-Aldrich, St. Louis, MO, USA). After homogenization, samples were centrifuged at 12000 × g for 20 min, supernatants were collected, and protein levels of each supernatant were quantified. Electrophoresis on 8-16% gradient SDS-PAGE was performed in order to resolve the 20 μg of proteins from the ventral midbrain tissues. The proteins were then transferred to nitrocellulose membranes. The membranes were blocked with blocking solution (Tris-buffered saline containing 5% non-fat dry milk and 0.1% Tween-20) for 1 h and incubated at 4 °C overnight with anti-α-synuclein (1:1000; Sigma S5566), anti-α-synuclein (1:1000; BD Biosciences), or anti-grp78 (1:500; Santa Cruz; sc-1050) antibodies, followed by HRP-conjugated secondary antibody (1:5000; GE Healthcare) for 1 h at RT. Finally, the membranes were re-probed with HRP-conjugated β-actin antibody (1:50,000; Sigma-Aldrich, St. Louis, MO, USA) after the blots were stripped.
Anti α synuclein
Manufactured by Merck Group
Sourced in United States
Anti-α-synuclein is a laboratory product manufactured by Merck Group. It is an antibody that specifically binds to the alpha-synuclein protein. This protein is a key component in the study of neurodegenerative disorders such as Parkinson's disease.
Automatically generated - may contain errors
Lab products found in correlation
Anti α synuclein, by BD (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti lamp2, by Santa Cruz Biotechnology (2 mentions)
Anti β tubulin, by Merck Group (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Anti rab4a, by Santa Cruz Biotechnology (2 mentions)
Anti rab5a, by Merck Group (2 mentions)
Anti rab7, by Merck Group (2 mentions)
Anti rabaptin5, by Santa Cruz Biotechnology (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Hrp conjugated β actin antibody, by Merck Group (1 mentions)
Phosphate inhibitor mixture 1 and 2, by Merck Group (1 mentions)
Anti grp78, by Santa Cruz Biotechnology (1 mentions)
Diax 900 homogenizer, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facs permeabilizing solution, by BD (1 mentions)
8 protocols using anti α synuclein
1
Ventral Midbrain Protein Extraction and Analysis
2
Age-Dependent Regulation of Vesicular Trafficking Proteins in Entorhinal Cortex
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Mice were anesthetized with ketamine (50 g/kg)/xylazine (5 mg/kg) and brains were rapidly removed over ice. Brains of 12 (n=4) and 24 (n=5) month old mice were snap frozen and stored at −80°C before biochemical analyses. Entorhinal cortices were dissected out from fresh brains of 12 (n=4) and 24 (n=4) month old mice and stored at −80°C. Western blot immunolabeling was performed as previously described52 (link). Primary antibodies used were: anti-LAMP-2 (1:1000), anti-Rab4a (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA; Cat # SC-312), anti-Rab5a (1:1000), anti-Rab7 (1:1000; Sigma, St. Louis, MO; Cat # R8779), anti-Rabaptin5 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA; Cat # SC-6162), anti-LC3 (1:1000), anti-Cat D (1:1000), C1/6.1 (1:1000)48 (link), anti-mTOR and anti-AKT (phosphorylated and total protein) (1:1000, Cell Signaling Technology, Inc., Danvers, MA; Cat # 2983S and Cat # 9272S respectively), anti-α-synuclein (1:1000; Sigma, St. Louis, MO; Cat # S3062), and anti-β-tubulin (1:10000; Sigma, St. Louis, MO; Cat # T8535). Secondary antibodies used were: HRP conjugated anti-rabbit and mouse antibodies (1:5000; GE Healthcare, Pittsburgh, PA). The protein bands were scanned, optical density was calculated using the Image J, and the ratio of protein intensity to β-tubulin in the same lane was calculated. Western blot analyses were repeated 3 times.
3
Intracellular Analysis of α-Synuclein in T Cells
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Intracellular phenotyping of purified T cells was performed as previously described.37 (link) Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with FACS permeabilizing solution (BD BioSciences, San Jose, CA, USA; 340973), washed and stained. Alpha-synuclein expression was detected using anti-α-synuclein (1 μg × 106 cells, Sigma, St. Louis, MO, USA; S5566) monoclonal antibody (mAb) and FITC-conjugated anti-mouse IgG (Fab specific, Sigma, F2653) as a secondary antibody. An appropriate isotype control antibody (Sigma, M5409) was used. Acquisition was performed on a FACSCalibur (BD BioSciences) and data were analyzed using the CellQuest Pro software (BD BioSciences).
4
Protein Aggregation Assay with Antibodies
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The following reagents were purchased from the indicated companies: MG-132 (474790; Calbiochem, Danvers, MA, USA) and Hoechst 33258 (H-3569; Molecular Probes, Eugene, OR, USA). The following antibodies were used in this study: anti-USP10 (A300-901A; Bethyl Laboratories, Montgomery, TX, USA; HPA006731; Sigma-Aldrich, St. Louis, MO, USA), anti-ubiquitin (sc-8017; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p62 (PM045; MBL, Nagoya, Japan, GP62-C; PROGEN, Heidelberg, Germany), anti-G3BP1 (611127; BD Transduction Laboratories, San Jose, CA), anti-G3BP2 (A302-040; Bethyl Laboratories), anti-PABP (ab21060; Abcam, Cambridge, GB), anti-HDAC6 (sc-11420; Santa Cruz Biotechnology), anti-FLAG (M2 Monoclonal Antibody; Sigma-Aldrich), anti-GFP (sc-9996; Santa Cruz Biotechnology), anti-lamin B1 (sc-374015; Santa Cruz Biotechnology), anti-α-synuclein (S5566; Sigma-Aldrich), anti-phosphorylated α-synuclein (015-25191; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), anti-β-actin (sc-47778; Santa Cruz Biotechnology), anti-HA (2367S; Cell Signaling, Beverly, MA, USA) and anti-α-tubulin (CP06 Oncogene Research Products, Boston, MA, USA).
5
Protein Extraction and Western Blot Analysis
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Total protein extracts were prepared from samples taken at 2 OD600 (optical density at 600 nm) units in sample buffer containing 50 mM Tris, pH 8, 2% SDS, 0.1% bromophenol blue, 10% glycerol. Antibodies used were: Anti-α-synuclein (Sigma, S3062, 1:1000), Anti-ADH2 (Chemicon, AB1202, 1:10000) and mouse Anti-rabbit complexed with HRP (Santa Cruz, 1:1000). Membranes were blocked in TBS-T (0.05% Tween-20) with 5% BSA. Antibodies were diluted in TBS-T (0.05% Tween-20) with 5% BSA. Primary antibodies were incubated overnight at 4°C. The secondary antibody was incubated for 1 h at room temperature.
6
Age-Dependent Regulation of Vesicular Trafficking Proteins in Entorhinal Cortex
7
Stereological Analysis of Dopaminergic Neurons
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The caudal parts of the brains were rapidly removed, post-fixed in cold 4% paraformaldehyde for 7 days, and cryoprotected in a 20% sucrose solution in phosphate buffered saline (PBS) until they sank. The brains were then cut on a freezing microtome into 30 μm frontal sections according to stereological rules. Each 6th section was sampled into a series of sections that covered all lengths of the SN and adjacent series were stained as previously described [28 (link)]. Free-floating sections were incubated for 48 h at 4 °C in a primary antibody (anti-tyrosine hydroxylase (TH), 1: 3,000; anti-α-synuclein, 1:4,000; anti-synphilin-1, 1:3,000; (all from Chemicon Int.; Millipore, USA) and anti—CD11b, 1:500 AbD Serotec, UK, anti-Iba1, 1:2,000, Wako, Japan), rinsed in PBS, then incubated for 30 min in secondary antibodies (anti mouse or anti rabbit biotinylated, 1:200, Vector Laboratories, UK) and processed using an ABC-peroxidase kit (Vector Laboratories, UK) and 3,3ʹ-diaminobenzidine as a chromogen. The stained sections were mounted on slides, dried, stained with 1% cresyl violet (CV) and covered. TH immunoreactive (TH + /CV +) dopaminergic neurons and non-DA neurons (TH −/CV +) were counted stereologically in the SN and ventral tegmental area (VTA).
8
Immunohistochemical Analysis of Synuclein Pathology
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The procedure for immunohistochemical analysis has been described elsewhere60 (link),93 (link). Briefly, blind-coded sagittal sections were incubated with the following primary antibodies; anti-α-synuclein(Syn1, #610787, BD bioscience, San Diego, CA, 1:250), anti-α-synuclein (Syn211, # 36-008), anti-NeuN(#MAB377, 1:1000), anti-GFAP (#MAB3402, 1:1000), anti-TH (# AB152, Millipore, County Cork, Ireland, 1:1000), and anti-Iba1 (#019-19741, Wako, Richmond, VA, 1:1000). To detect protease K (PK) resistant α-synuclein aggregates, the sections were pretreated with PK (10 µg/ml) for 8 minutes prior to incubating with anti-α-synuclein antibody94 (link). After overnight incubation at 4 °C, the sections were incubated with biotinylated secondary antibodies and subsequently detected utilizing an ABC staining kit (both from Vector Laboratories, Burlingame, PA). All sections were imaged by an Olympus BX41 microscope, and the immunoreactivity levels were determined by utilizing ImageJ (NIH). To determine α-synuclein pathology, neurodegeneration, microgliosis, and astrogliosis, the optical density of α-synuclein and GFAP per field (230 mm×184 mm) and the numbers of α-synuclein-positive, NeuN-positive, TH-positive fibers, Iba1-positive cells per indicated field were analyzed using Image Quant 1.43 program (NIH).
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