The nodules were fixed on ice in a freshly prepared solution of 3% paraformaldehyde (Sigma-Aldrich, Burlington, MA, USA) in MTSB buffer of 1/3 strength (50 mM PIPES (pH 6.9) (Amresco, Boise, ID, USA); 5 mM MgSO4x7H2O; 5 mM ethylene glycol-bis (β-aminoethyl ether) -N, N, N′, N′-tetraacetic acid (Sigma-Aldrich, Burlington, MA, USA) with the addition of 0.25% glutaraldehyde (solution Grade I, 25% in H2O. Sigma-Aldrich, Burlington, MA, USA), 0.3% Twin-20 (Amresco, Boise, ID, USA), 0.3% Triton-X-100 (Amresco, Boise, ID, USA). For optimal penetration of the fixative, the air from the tissue was evacuated 3 times for 7 min at 0.9 bar using a ME 1 vacuum pump (Vacuubrand, Wertheim, Germany) and left overnight at +4 °C. Then, the material was washed with PBS buffer (0.137 M NaCl, 0.0027 KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, pH 7.4). Fixed nodules were embedded in 2.5% agarose (Agarose, 1/4 MTSB) and sectioned on a vibrotome (TED PELLA, Inc., Redding, CA, USA). Sections were then freed from agarose and stained with propidium iodide.
Ethyleneglycol bis β aminoethyl ether n n n n tetraacetic acid
Manufactured by Merck Group
Sourced in United States
Ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid is a chemical compound that functions as a chelating agent. It is capable of binding and sequestering metal ions.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Triton x 100, by Avantor (2 mentions)
Pipes, by Avantor (2 mentions)
Twin 20, by Avantor (2 mentions)
Sodium deoxycholate, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Pepstatin, by Merck Group (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Caspase 9, by Abcam (1 mentions)
2 amino 2 hydroxymethyl propane 1 3 diol, by Merck Group (1 mentions)
Catalase, by Abcam (1 mentions)
Trigonelline, by Merck Group (1 mentions)
Antipain, by Merck Group (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse and rabbit igg, by Abcam (1 mentions)
Caspase 3, by BD (1 mentions)
7 protocols using ethyleneglycol bis β aminoethyl ether n n n n tetraacetic acid
1
Fixation and Sectioning of Plant Nodules
2
Pisum sativum Antioxidant Enzyme Assay
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The voucher specimens (Pisum sativum L. var. saccharatum Poir: CMU-104-PS-003) were deposited in Herbarium of College of Pharmacy, China Medical University, Taichung, Taiwan. Antipain, aprotinin, dithiothreitol, ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, leupeptin, Nonidet P-40, pepstatin, phenylmethylsulfonyl fluoride, sodium deoxycholate, trigonelline, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) were purchased from Sigma Chemical Company (St. Louis, MO). Antibodies to various proteins were obtained from the following sources: β-actin antibody was purchased from Sigma Chemical Company; Caspase-9, catalase, p38 (pThr180/Tyr182), and Raf (pSer259) were purchased from Abcam (Cambridge, MA); Mn-SOD and Cu/Zn-SOD were from Calbiochem (San Diego, CA); ERK (pThr202/Tyr204) was from ThermoFisher Scientific, Inc. (Waltham, MA); Nrf2 (pSer40) was from GeneTex, Inc. (Irvine, CA); Caspase-3 and protein kinase Cα (PKCα) from BD Biosciences (San Jose, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and -rabbit IgG were from Abcam.
3
Preparation and Characterization of PMX Nanoparticles
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The PMX disodium hemipentahydrate was obtained from Shipla Medicare (Karnataka, India). Caprylocaproyl macrogol-8-glyceride (Labrasol) was provided by Gattefossé (Saint Priest, France). Polyoxyethylene (160) polyoxypropylene (30) glycol (P188) was purchased from BASF (Ludwigshafen, Germany). Chlorpromazine, methyl-β-cyclodextrin (MβCD), brefeldin A, genistein, actinomycin D (Act D), cyclosporine A, ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), DOCA, and 4-[N-(2,4-diamino-6-pterinidinylmethyl)-N-methylamino]benzoic acid hemihydrochloride hydrate (DAMPA) were obtained from Sigma-Aldrich Inc. (St. Louis, MO). Anti-mouse PD-1 antibody (anti-PD1) was purchased from Bio X Cell (West Lebanon, NH). Solvents for high-performance liquid chromatography (HPLC) were obtained from Merck Millipore (Billerica, MA) and Thermo Fisher Scientific Inc. (Waltham, MA).
4
Neurochemical Signaling Protocol
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Methyl sulfoxide (DMSO), PTX, K-gluconate, Lucifer yellow, K2-ATP, Na3-GTP, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (Na-TES), nicotinamide adenine dinucleotide phosphate (NADP+), glutamate dehydrogenase, sodium dodecyl sulfate (SDS), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), Triton X-100, and 0.01 M PBS were purchased from Sigma. [S-(R*,R*)]-[3-[[1-(3,4-Dichlorophenyl)ethyl]amino]-2-hydroxypropyl] (cyclohexylmet-hyl) phosphinic acid (CGP54626) and (3-aminopropyl) ethylphosphinic acid hydrochloride (CGP36216) were purchased from Tocris. Morphine was from Shenyang No. 1 Pharmaceutical Factory, China. Percoll was purchased from Amersham Biosciences Corporation. Other AR grade reagents were from Shanghai Chemical Plant. PTX or CGP54626 was dissolved in DMSO and others were dissolved in ddH2O. When DMSO was used as the vehicle, drugs were initially dissolved in 100% DMSO and then diluted into ASFC at a final DMSO concentration of less than 0.5%, which had no detectable effects on the parameters we observed.
5
Chondrocyte Differentiation Pathway Reagents
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Sodium phosphate (NaH2PO4 and Na2HPO4), ascorbic acid, alizarin red S, cetylpyridinium chloride, silver nitrate, ethylenediaminetetraacetic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, NP-40, and sodium deoxycholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin-streptomycin antibiotic mixture and FBS were purchased from Life Technologies (Grand Island, NY, USA). DMEM and Hanks’ balanced salt solution (HBSS) were purchased from Welgene (Daegu, Korea). Phosphatase- and protease-inhibitor cocktails were purchased from Roche Diagnostics (Manheim, Germany). Antibodies specific for Bax, Bcl-2, and α-SMA were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Runx2 and Pit-1 were obtained from Proteintech Group (Chicago, IL, USA). Anti-β-actin and anti-SM22α were obtained from Sigma-Aldrich and Abcam (Cambridge, MA, USA), respectively. A polyclonal APE1/Ref-1 antibody generated by immunizing rabbits with recombinant human APE1/Ref-1 was prepared in our laboratory [26 (link)].
6
Kinase Activity Assay Protocol
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Original samples from the PKIS were obtained from GSK. The compounds GW301784X, GW837331X, GW406108X, GSK2358994A, GSK846226A and GW429374A were synthesised according to previously published methods [20–25 (link)]. Protein kinases (ULK1, VPS34 and AMPK α1β2ɣ1) and SAMS peptide were purchased from MRC PPU Reagents and Services, Dundee, Scotland. ADP Hunter™ Plus kits and ultra-pure ATP Gold were from Discoverx. SBI-0206965 and VPS34-IN1 were obtained from Selleck Chemicals. MRT68921 was provided by LifeArc (formerly MRC-Technology). Trizma® hydrochloride, Trizma® base, Tris(2-carboxyethyl)phosphine (TCEP), Pluronic® F-127, sodium chloride (NaCl), magnesium chloride (MgCl2), manganese chloride (MnCl2), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimethyl sulfoxide (DMSO) and bovine myelin basic protein (MBP) substrate were from Sigma–Aldrich. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tween-20 and PI:PS lipid kinase substrate were from Thermo Fisher Scientific. Reagents used for Western blot and immunofluorescence were published previously [26 (link)].
7
Fixation and Preparation of Nodules
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The nodules were fixed in a freshly prepared solution of 2% paraformaldehyde (Sigma-Aldrich, MO, USA) in 1/3 of the concentration of MTSB buffer (50 mM PIPES (pH 6.9) (Amresco, OH, USA); 5 mM MgSO4 × 7H2O; 5 mM ethylene glycol-bis (β-aminoethyl ether)—N, N, N‘, N’-tetraacetic acid (Sigma, MO, USA) with the addition of 0.25% glutaraldehyde (solution Grade I, 25% in H2O. Sigma-Aldrich, MO, USA), 0,3% Twin-20 (Amresco, OH, USA), 0,3% Triton-X-100 (Amresco, OH, USA). For optimal penetration of the fixative, the air from the tissue was evacuated three times for 7 min at 0.9 bar using a ME 1 vacuum pump (Vacuubrand, Germany) and left overnight at +4 °C. Then, the material was washed with PBS buffer (0.137 M NaCl, 0.0027 KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, pH 7.4).
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