Total protein concentration in each sample was determined by a bicinchoninic acid colorimetric assay (Fisher Cat#23223 and 23224), using bovine serum albumin as a standard (Thermo Fisher Cat#23210). Protein was resolved on 5–20% gradient polyacrylamide gels using equal protein loading. Proteins were transferred to 0.2 um nitrocellulose membranes and detected with primary antibodies (Syn9027, CNDR, 1:20,000), pS129 α-synuclein (Abcam Cat#ab168381, 1:1000), GCase (Sigma-Aldrich Cat#G4171, 1:1,000), GCase (Santa Cruz Biotech Cat#sc-166407, 1:200), GFAP (Agilent Cat# Z0334, 1:5,000) or GAPDH (2-RGM2, Advanced Immunological, 1:5000). Primary antibodies were detected using IRDye 800 (Li-cor 925–32210) or IRDye 680 (Li-cor 925–68071) secondary antibodies, scanned on a Li-cor Odyssey Imaging System and analyzed using Image Studio software. Values obtained from this program were normalized to GAPDH or GFAP, and were further normalized to the mean control values.
Ps129 α synuclein
Manufactured by Abcam
PS129-α-synuclein is a recombinant protein product that represents the post-translationally modified form of the α-synuclein protein, where serine 129 is phosphorylated. This product can be used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Gcase, by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
β actin, by AbFrontier (1 mentions)
Chemidoc trs, by Bio-Rad (1 mentions)
Txnip, by Thermo Fisher Scientific (1 mentions)
Phospho p38, by Thermo Fisher Scientific (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Proteinase inhibitor mixture, by Roche (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Zen analysis software package, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Eclipse ti, by Nikon (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Shandon cryotome, by Thermo Fisher Scientific (1 mentions)
4 protocols using ps129 α synuclein
1
Western Blot Quantification of Neurological Proteins
2
Molecular Analysis of Dopaminergic Organoids
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Organoids were dissociated by a homogenizer and washed with 1× PBS. Homogenized organoids were extracted in lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl in 50 mmol/L Tris [pH 8.0], Sigma-Aldrich; and 1× proteinase inhibitor mixture, Roche). The extracted protein was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was probed with the following primary antibodies: NURR1 (1:200, Santa Cruz, sc-991), VMAT2 (1:1,000, Abcam, AB1598P), PITX3 (1:1,000, Invitrogen, 382850), pS129-α-synuclein (1:500, Abcam, AB9850), cleaved caspase-3 (1:500, Cell Signaling, 9661s), TXNIP (1:500, Thermo, 40-3700), Phospho-ERK1/2 (1:1,000, Cell Signaling, 4370s), ERK1/2 (1:1,000, Cell Signaling, 4695s), Phospho-p38 (1:500, Thermo, MA5-15177), p38 (1:500, Antibodies online, abin2957701), and β-actin (1:1,000, AbFrontier, LF-PA0207). Representative images are shown of western blots performed using Chemidoc TRS+ with Image Lab software (Bio-Rad).
3
Quantifying Synuclein Neuropathology in Mouse Brains
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Mouse brains were fixed in 10% (vol/vol) formalin, processed, embedded, and sectioned as previously described [25 (link)]. Brains were cut into four sections prior to processing through graded alcohols, clearing with xylene, infiltrating with paraffin, and embedding. After deparaffinization, sections were exposed to heat-mediated antigen retrieval with citrate buffer (0.1 M, pH 6) for 20 min. Slides were stained overnight at room temperature after blocking in 10% (vol/vol) normal goat serum using EP1536Y (pS129 α-synuclein; 1:1,000; Abcam), p62 (Anti-SQSTM1; 1:1,000; Abcam), and glial fibrillary acidic protein (GFAP; 1:500; Abcam) primary antibodies. Secondary antibodies conjugated to AlexaFluor 488, 568, or 647 (Thermo Fisher) were used to detect immunolabeling.
Slides were imaged using the Zeiss AxioScan.Z1. Digital images were analyzed using the Zen Analysis software package (Zeiss). To quantify α-synuclein neuropathology, a pixel intensity threshold was determined using a positive control slide and was then applied to all slides. Regions of interest were drawn around the HC, Thal, HTH, midbrain (Mid), and pons. The percentage of pixels positive for staining in each region was determined.
Slides were imaged using the Zeiss AxioScan.Z1. Digital images were analyzed using the Zen Analysis software package (Zeiss). To quantify α-synuclein neuropathology, a pixel intensity threshold was determined using a positive control slide and was then applied to all slides. Regions of interest were drawn around the HC, Thal, HTH, midbrain (Mid), and pons. The percentage of pixels positive for staining in each region was determined.
4
Immunohistochemical Analysis of Organoid Sections
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Organoids were washed with 1× PBS before being fixed in 4% paraformaldehyde in PBS and cryoprotected in a 30% sucrose solution overnight. Frozen organoids were sectioned at 15–20 μm using a Thermo Shandon cryotome and collected on glass microscope slides. Sections were immunostained according to standard protocols using the following primary antibodies: OTX2 (Abcam, AB21990), FOXA2 (Abcam, AB108422), NGN2 (Millipore, AB5682), MASH1 (BD Biosciences, 556604), TUJ1 (Sigma, T2200), MAP2 (Cell Signaling Technologies, 4542s), TH (Pel-Freez Biologicals, P60101-150), VMAT2 (Abcam, AB1598P), DAT (Millipore, MAB369), PITX3 (Invitrogen, 382850), AADC (Abcam, AB211535), NURR1 (Santa Cruz, sc-991), GIRK2 (Abcam, AB65096), cleaved caspase-3 (Cell Signaling, 9661s), pS129-α-synuclein (Abcam, AB9850), LAMP1 (Developmental Studies Hybridoma Bank), γH2AX (Cell Signaling, 9718s), LC3B (Cell Signaling, 3868s), PARKIN (Millipore, MAB5512), NRF2 (Cell Signaling, 12721s), and EEA1 (Millipore, 07-1820). Appropriate fluorescent secondary antibodies were obtained from Invitrogen. Next, sections were treated with 6-diamidino-2-phenylindole (Invitrogen) and mounted in Fluoromount-G mounting medium. Representative images were captured using a Nikon Eclipse Ti microscope and a confocal laser scanning microscope (Zeiss, LSM800).
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