The neurovirulent RP-9 strain of JEV was used for both in vitro and in vivo studies and was propagated in mosquito C6/36 cells as described (Chen et al., 1996 (link)). Viruses were titrated by plaque-forming assay in baby hamster kidney BHK-21 cells (ATCC: CCL-10). Dopaminergic human neuroblastoma BE(2)C cells (ATCC CRL-2268) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum. Primary antibodies included anti-JEV-NS3, anti-phospho-tyrosine hydroxylase (Cell Signaling, #2791), anti-tyrosine hydroxylase (Cell Signaling, #2792), anti-D2R (Santa Cruz Biotechnology, sc-9113), anti-D1R (Santa Cruz Biotechnology, sc-1434), anti-phospho-CaMKII (Thermo Scientific, 22B1), anti-integrin β3 (BD Biosciences, 611140), anti-vimentin (Sigma, V6389), anti-β-actin (Chemicon), and anti-α-tubulin (Sigma–Aldrich).
Anti tyrosine hydroxylase
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-tyrosine hydroxylase is a primary antibody that binds to and detects the tyrosine hydroxylase protein. Tyrosine hydroxylase is a key enzyme involved in the biosynthesis of the neurotransmitters dopamine, norepinephrine, and epinephrine.
Automatically generated - may contain errors
Lab products found in correlation
Anti α tubulin, by Merck Group (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti d2r, by Santa Cruz Biotechnology (1 mentions)
Anti d1r, by Santa Cruz Biotechnology (1 mentions)
Anti phospho camkii, by Thermo Fisher Scientific (1 mentions)
Anti integrin β3, by BD (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg hrp, by Merck Group (1 mentions)
Anti rabbit igg hrp, by Merck Group (1 mentions)
Anti map2, by Cell Signaling Technology (1 mentions)
Anti β1 integrin antibody, by BD (1 mentions)
Anti ha antibody, by Santa Cruz Biotechnology (1 mentions)
Anti at8, by Thermo Fisher Scientific (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
Anti iba1, by Abcam (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Homovanillic acid, by Merck Group (1 mentions)
Doxycycline, by MedChemExpress (1 mentions)
Chloroquine, by Merck Group (1 mentions)
11 protocols using anti tyrosine hydroxylase
1
Propagation and Characterization of JEV
2
Antibodies Used in Western Blot Analysis
3
Immunohistochemical Analysis of Mouse Brain
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Brain specimens were collected from mice in saline and MPTP groups. Coronal sections (40um) containing the SN, cortex, CA3 area of hippocampus, amygdala area and VTA were cut using a cryostat microtome. Brain sections were incubated with an anti-p-α-Synuclein (#23706, Cell Signaling Technology, Danvers, MA, USA), anti-Tyrosine Hydroxylase (#58844, Cell Signaling Technology, Danvers, MA, USA), anti- AT8 (#MN1020, Thermo Fisher Scientific, MA, USA) or anti-Iba1 (#ab178846, Abcam plc, Cambridge, UK). Second Alexa Fluor 568 or 488 conjugated antibody (#A-11031, #A-11001, Thermo Fisher Scientific, MA, USA) were incubated for 1 h followed by mounting in VECTASHIELD Antifade Mounting Medium containing DAPI (H-1200, Vector Laboratories, CA, USA). Images were obtained using a confocal LSM 880 microscope (Zeiss, Wurttemberg, Germany).
4
Biochemical Assays for Autophagy
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Harmol was purchased from Tokyo Chemical Industry. Doxycycline was purchased from MedChemExpress. Dorsomorphin dihydrochloride was purchased from Targetmol. G418 sulfate was purchased from Amresco. Dopamine hydrochloride, homovanillic acid, 3, 4-dihydroxyphenylacetic acid, tri-sodium citrate dihydrate, citric acid, chloroquine, anti-Flag M2, and anti-α-synuclein antibodies were purchased from Sigma-Aldrich. anti-TFEB antibody was purchased from Proteintech. Anti-tyrosine hydroxylase, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-ULK1 (Ser757), anti-phospho-ULK1 (Ser317), anti-phospho-ULK1 (Ser555), anti-ULK1, anti-phospho-AMPKa (Thr172), anti-AMPKa, anti-LAMP1, anti-SQSTM1/p62, anti-TFEB, and anti-CTSD/cathepsin D antibodies were purchased from Cell Signaling Technology. Anti-phospho-α-synuclein and anti-LC3B antibodies were purchased from Abcam. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Beyotime Biotechnology.
5
Western Blotting and Flow Cytometry Analysis
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For Western blotting, we used anti-UCP1 (U6382, 1:1000) and anti–β-Actin (A3854, 1:40,000) from MilliporeSigma; anti-PGC1α (ab54481, 1:1000), anti-FGF21 (ab64857, 1:1000) from Abcam; anti-C/EBPβ (sc7962, 1:1000) from Santa Cruz Biotechnology; and anti-Nr1d1 (Rev-Erbα) (13418, 1:1000), anti–Tyrosine Hydroxylase (2792, 1:1000), and anti-Phospho-(Ser/Thr) PKA Substrate (9621, 1:1000) from Cell Signaling Technology. For flow cytometry, we used anti-F4/80 (123110, 1:200), anti-CD206 (141704, 1:200), anti-CD11b (101228, 1:200), and anti-CD11c (117310, 1:200) from BioLegend.
6
MPTP-Induced Parkinson's Disease Model
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MPTP was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 5-Bromo-2-deoxyuridine (BrdU) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium pentobarbital was purchased from Hanlim Pharm Co., Ltd. (Seoul, South Korea). Biotinylated secondary antibodies (1:200, Cat# BA-1000) and fluorochrome-conjugated secondary antibodies (1:500, Cat# DI-3094, DI-1094, DI-2594), diaminobenzidine tetrahydrochloride, and antifade reagent were obtained from Vector Laboratories (Burlingame, CA, USA). The following primary antibodies were used in this study: anti-tyrosine hydroxylase (1:2000, Cat# 58844), anti-Ki67 (1:500, Cat# 9129), anti-doublecortin (1: 500, Cat# 4604) and anti-p-AMPK (1:1000, Cat# 2535) antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA) and anti-BDNF (1:1000, Cat# ab203573) and anti-BrdU (1:250, Cat# ab6326) from Abcam (Cambridge, UK).
7
Monocrotaline-Induced Pulmonary Hypertension Model
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Monocrotaline (MCT, NO. M0418–500), NE (NO.M8550) and prazosin (Pra, NO.MT1050) were provided by Mengbo (Chongqing, China). U0126 (NO. NY-12031) and Cell Counting Kit-8 (CCK-8, NO. HY-K0301) were obtained from MedChemExpress (MCE, NJ, USA). The NE Elisa Kit (NO. 2907A) was obtained from MB-Biology (Jiangsu, China). The anti-tyrosine hydroxylase (TH, NO.#13,106), ERK-1/2 (NO.#4695) and p-ERK-1/2 (NO.#9101) antibodies were provided by Cell Signaling Technology (Beverly, MA, USA). The anti-α-smooth muscle actin (α-SMA) (NO. ab5694), CD3 (NO. ab16669), cyclin A2 (NO. ab181591), and cyclin D1 (NO. ab134175) antibodies were purchased from Abcam (Cambridge, UK). The proliferating cell nuclear antigen (PCNA, NO. 10205–2-AP), β-actin (No.20536–1-AP), and GAPDH (No.10494–1-AP) antibodies and the secondary antibodies (NO.SA00001–10) were obtained from Proteintech (Wuhan, Hubei, China). All reagents were obtained from common commercial sources.
8
Western Blot Analysis of Neurological Proteins
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Tissues were homogenized in lysis buffer [Tris 50 mM pH 8, NaCl 150 mM, EDTA 10 mM, triton-x100 1%, Sodium deoxycholate 0.5%, cOmplete™ Protease Inhibitor Cocktail (Roche, Brazil), Phosphatase Inhibitor Cocktail set II (Millipore, Brazil)] and post-nuclear supernatant was collected after the centrifugation at 3,000 × g for 5 min. Following SDS-PAGE, proteins were transferred to PVDF membrane and the protein of interest was detected using primary antibodies: SAF32 for PrPC (Cayman Chemical, USA, Cat #189720); anti- α-synuclein (Cell Signaling Technology, USA, Cat # 2642); anti- tyrosine hydroxylase (TH) (Cell Signaling Technology, USA, Cat # 27925); anti- DAT (EDM Millipore, USA, Cat # MAB369); anti- GAPDH (Cell Signaling Technology, USA, Cat #2118). The signals were revealed with HRP-conjugated secondary antibodies and Luminata™ Forte Western HRP Substrate (Millipore, USA, Cat#WBLUF0500). Digital images of membranes were acquired using Alliance Mini gel documentation system (UVITEC, UK). Band intensity was quantified using software UVIband (UVITEC, UK). The intensity of the protein of interest was normalized with the GAPDH band and data were presented as percentage of mean of control group.
9
Immunofluorescence Staining of Cells
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Cells were grown on chamber slides and coating cover slides and fixed with 3.7% paraformaldehyde in PBS. The membrane was permeabilized by treating cells for 2 min with 0.1% Triton X-100 in PBS. The cells were then placed in blocking solution (5% FBS in PBS) at room temperature. Cells were incubated with primary antibodies for 1 h at room temperature. After washing, they were incubated with the Alexa Flour 488 (excitation/emission = 495/519 nm, green, Invitrogen, CA, USA) and Alexa Flour 594 (excitation/emission = 590/617 nm, red, Invitrogen, CA, USA) for 30 min at room temperature. Cells were counterstained with Hoechst 33342 (excitation/emission = 330–380 nm/460 nm, ImmunoChemistry, MN, USA). Slides were mounted using ProLong® Gold antifade reagent (Molecular Probes® by Life Technologies™, CA, USA). Primary antibodies utilized are the following: anti-pNF-κB, anti-pSTAT3, and anti-tyrosine hydroxylase (TH) (from Cell Signaling Technology). Immunolabeling was examined using an Eclipse Ti-U and confocal microscope (Nikon, Tokyo, Japan).
10
Parkinson's Disease Cell Model Protocol
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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 100 units/mL penicillin and 100 mg/mL streptomycin are purchased from Gibco (Thermo Fisher ScientificWaltham, MA, USA). 1-Methyl-4-pheynl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride, 1-Methyl-4-phenylpyridinium (MPP+) iodide, 3-methyladenine (3-MA) and ropinirole were purchased from Sigma Chemical Co (St. Louis, MO, USA). Wortmannin (Wort) and bafilomycin A1 (Baf) are from Abcam (MA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-AMPK, anti-phosphorylated AMPK (p-AMPK), anti-ULK, anti-phosphorylated ULK555 (p-ULK555), anti-Erk, anti-phosphorylated Erk (p-Erk), anti-mTOR, anti- phosphorylated mTOR (p-mTOR), anti-LC3B, anti-tyrosine hydroxylase (TH), anti-α-synuclein primary antibodies sourced from rabbit, anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody, the Erk inhibitor U0126, the AMPK siRNA, AMPK siRNA control were purchased from Cell Signaling Technology (Boston, MA, USA). An autophagy Tandem Sensor RFP-GFP-LC3B kit was from Thermo Fisher Scientific. MTT assay kit Z-Cytox was from DAEILLAB Co, Ltd (Seoul, Korea). Dopamine ELISA kit was from Abnova (Taipei City, Taiwan) and the MAO-B assay kit was supplied by Promega (Woods Hollow Road Madison, WI, USA). All other chemicals were of the highest grade and were found from commercial resources.
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