Double-fluorescence immunohistochemistry was performed on coronally sectioned 4% paraformaldehyde (PFA) fixed brain tissue from wild-type ad libitum fed mice. To detect immunofluorescence, sections (40 μm) were incubated with the following antibodies (NUCB2/nesfatin-1: #H-003-22, 1:1000, Phoenix Pharmaceuticals; tyrosine hydroxylase: #T1299, 1:500, Sigma Aldrich; calretinin: #AB1550, 1:1000, Millipore; GAD67: #MAB5406, 1:1000, Millipore) followed by species-specific Alexa 488, 633 and 647 secondary antibodies. Fluorescence images were acquired on a Leica SP5 confocal microscope and analyzed with Image J software (NIH). For details, see Supplementary Materials and Methods .
Tyrosine hydroxylase
Manufactured by Merck Group
Sourced in United States, United Kingdom
Tyrosine Hydroxylase is a laboratory enzyme that catalyzes the conversion of the amino acid tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). It is a key enzyme in the biosynthesis of the neurotransmitters dopamine, norepinephrine, and epinephrine.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (8 mentions)
Neuronal nuclei (neun), by Merck Group (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
β tubulin, by Merck Group (3 mentions)
β actin, by Abcam (3 mentions)
Cryostat, by Leica camera (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
Drp1 d6c7, by Cell Signaling Technology (2 mentions)
Tom20 d8t4n, by Cell Signaling Technology (2 mentions)
Disposable pellet mixers with pestle motor, by Avantor (2 mentions)
Dopamine d2 receptor, by Merck Group (2 mentions)
Phospho tau ser202 thr205 at8, by Thermo Fisher Scientific (2 mentions)
Mitofusin 2 d2d10, by Cell Signaling Technology (2 mentions)
Ha tag c29f4, by Cell Signaling Technology (2 mentions)
α synuclein, by BD (2 mentions)
Alexa fluor 488 cy3, by Jackson ImmunoResearch (2 mentions)
Upright widefield microscope, by Nikon (2 mentions)
Fluoromount, by Southern Biotech (2 mentions)
46 protocols using tyrosine hydroxylase
1
Dual-Labeled Immunohistochemistry of Mouse Brain
2
Protein Extraction and Western Blot Analysis of Neural Tissues
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Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0 °C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500 μl, 5 μl aprotinin, PMSF 50 μl, Na3VO4 100 μl and NaF 20 μl in 4.32 ml PBS. Extracts were clarified by centrifugation at 4 °C (13,200g for 20 min). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE [45 (link)]. The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), α-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
3
Hair Dye Formulation Protocol
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Dopamine hydrochloride was purchased from Inno-chem Co., Ltd. Tyrosine hydroxylase was purchased from Sigma-Aldrich (China). Iron chloride hexahydrate (FeCl3·6H2O), iron sulfate heptahydrate (FeSO4·7H2O) and copper sulfate pentahydrate (CuSO4·5H2O) were purchased from Aladdin Co., Ltd. To test the hair dyeing ability of our method, grey hair samples were purchased from Narcissus Salon. BALB/c mice were purchased from the Institutional Animal Care and Shanghai Slaccas Laboratory Animals Co., Ltd.
4
Protein Extraction and Analysis from Brain Tissues
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Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0°C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500μl, 5μl aprotinin, PMSF 50μl, Na 3 VO 4 100μl and NaF 20μl in 4.32 ml PBS. Extracts were clari ed by centrifugation at 4°C (13,200 g for 20 minutes). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE (43) . The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), a-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies.
Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.
5
Tyrosine Hydroxylation of Y-IGF-1
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The Y-IGF-1 protein was diluted with PBS to 1 mg/mL. The tyrosine residues of Y-IGF-1 were hydroxylated using solutions of tyrosine hydroxylase (500 mg/mL, 500 mL) (Sigma-Aldrich, St. Louis, MO), ascorbic acid (250 mM, 500 mL), Y-IGF-1 (1 mg/mL, 500 mL), and PBS (500 mL, pH 7.2) at room temperature for 2 h, then regulated PH to 8.5.
6
Molecular Mechanisms of Neuroprotection
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First, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tyrosine hydroxylase (TH) was purchased from Merck Millipore (Bedford, MA, USA). Brain-derived neurotrophic factor (BDNF), cyclooxygenase-2 (COX-2), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome c, mitochondrial transcription factor A (TFAM), and nuclear factor kappa B-p65 (NF-kB p65), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphorylated cyclic AMP response element binding (pCREB), cyclic-AMP response element binding (CREB), sirtuin-1 (SIRT-1), peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), cleaved caspase3, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and voltage-dependent anion channel (VDAC), were purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin purchased from Abcam (Cambridge, MA, USA) and all other chemicals used in this study were of analytical grade.
7
Immunocytochemistry of iPSC, NPC, and Neurons
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WT and HD iPSC/NPC colonies or 21-day old differentiated neurons were washed in PBS pH 7.2 and fixed in 4% paraformaldehyde. Blocking solution (1x PBST with 5% BSA) was added to cells for 60 min prior to antibody incubation. Cells were incubated in primary antibodies, OCT-4 (Santa Cruz 1:200), Nestin (Santa Cruz 1:200), MAP2 (BD Biosciences 1:200) βIII-tubulin (Biolegend 1:200), Tyrosine Hydroxylase (EMD Millipore 1:200), Rab4 (Abcam 1:200), HIP1 (Novus Biological, 1:200), DIC (Abcam, 1:200), or PolyQ (EMD Millipore, 1:200) for 16 h at 4 °C and appropriate secondary antibodies (AlexaFluor® 488 or 568, ThermoFisher) for 1 h at 25 °C. DAPI was used to stain nuclei. Cells were then imaged at 20x-40x (for iPSC and NPC) or 60x-100x (iNeurons). iNeurons were imaged on glass slide bottom dishes (In Vitro Scientific China, D29–14-1-N). As above, fixed images were taken at 100x using DAPI, FITC, TxRed, and Cy5 filters which were merged into a single RGB image to analyze co-localization noted as yellow or white puncta. Comprehensive list of reagents and antibodies used in this study can be found in Table S1 .
8
Hippocampus Protein Expression Analysis
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Hippocampal homogenates were determined using Bio-Rad DC protein assay kit (BIO-RAD, Hercules, CA, USA) according to the manufacturer’s protocol. For immunoblotting analysis, 30 μg hippocampus proteins were resolved over 12% Tris-glycine polyacrylamide gel and then transferred onto polyvinylidene fluoride membrane (Merck Millipore, Burlington, VT, USA). The blots were blocked using 5% nonfat dry milk, and these immunoblots were probed using the primary antibodies Tyrosine hydroxylase (EMD Millipore, Darmstadt, Germany), GAD-67 (Abcam, Cambridge, UK), CREB (Cell signaling, Danvers, MA, USA), phospho-CREB (EMD Millipore), GAPDH (Cell signaling) and secondary horseradish peroxidase conjugate (Amersham Life Science Inc., IL, USA). The proteins were detected by chemiluminescence using the ECL kit (Amersham Life Science). The relative density of the protein bands was scanned and quantified by ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).
9
Antibody Panel for Neuronal Markers
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The following primary antibodies were used: GFP (Cell Signaling Technology catalog number 2956, clone no. D5.1; dilution factor 1:1000,); Cdh5 (Santa Cruz Biotechnology catalog number sc9989, lot no. c1622, clone no. F-8; dilution factor 1:200), GAPDH (Cell Signaling Technology catalog number 2118, lot. No. 16, clone no. 14C10; dilution factor 1:2000,), NeuN (Millipore catalog number mab377, lot no. 382727; dilution factor 1:100), and Tyrosine Hydroxylase (EMD Millipore catalog number AB152, lot. No. 3845 [clone number is not available]; dilution factor 1:1000).
10
Imaging Dopaminergic Neurons in Drosophila Brains
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Adult brain dissections were performed as previously described [60 (link)]. Briefly, decapitated heads from 7‐week‐old flies were fixed in 4% formaldehyde in PTX (0.5% Triton X‐100, PBS) for 20 min, and washed five times for 2 min each in PTX. Dissected brains were fixed in 4% formaldehyde in PTX for 10 min, washed five times for 2 min each in PTX and then incubated with Tyrosine Hydroxylase (Merck, Kenilworth, NJ, USA, Cat #: Ab152, 1 : 400) and Cy™3 AffiniPure Goat Anti‐Rabbit IgG (H + L) (Jackson Immunoresearch, West Grove, PA, USA, Cat #: 111‐165‐003, 1 : 200) to detect dopaminergic neurons: Brains were then washed three times for 10 min each at room temperature and mounted in Vectashield for microscopy. Once coverslipped, Drosophila brains were imaged using the HC PL APO CS2 63x/1.4 objective using a zoom factor of 3.5 on the Leica TCS SP8 inverted confocal microscope. Z‐stack images were acquired using LAS X Navigator software.
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