24 hours following cocaine-induced CPP, animals were euthanized and their brains were immediately removed. PFC and NAc were microdissected, homogenized and analyzed for OS. The extent of lipid peroxidation in animal brain tissue was estimated colorimetrically using the thiobarbituric acid reactive substances (TBARS) method as we have previously described16 (link). Briefly, 0.1 ml aliquots of tissue homogenates were treated with 2 ml of (thiobarbituric acid (TBA)–TCA–HCl 1:1:1 ratio) reagent containing: thiobarbituric acid 0.37% (Sigma, St. Louis, USA) - TCA 15% (Baker, NJ, USA) - HCI 0.25 N (1:1:1 ratio). Samples were heated to 95 °C for 30 min, cooled on ice and centrifuged (12,000 rpm, 10 min). The clear supernatant fluids were measured at 535 nm with a microplate reader (Bio-TEK ELx, Winooski, VT, USA). A malonaldehyde bis (dimethyl acetal) (Sigma, St. Louis, USA) standard curve was prepared under the same experimental conditions. Calculations were made on the basis of standard curves and presented as µmol malondialdehyde (MDA)/g protein.
Thiobarbituric acid
Manufactured by Merck Group
Sourced in United States, Germany, India, United Kingdom, Sao Tome and Principe, Poland, Spain, Italy, Brazil, Macao, France, China, Czechia, Portugal, Canada, Mexico, Japan
Thiobarbituric acid is a chemical compound used in various laboratory applications. It is a white to pale yellow crystalline solid that is soluble in water and organic solvents. Thiobarbituric acid is commonly used as a reagent in analytical techniques to detect the presence of certain compounds, particularly those related to lipid peroxidation.
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Lab products found in correlation
Trichloroacetic acid, by Merck Group (241 mentions)
Gallic acid, by Merck Group (66 mentions)
Bovine serum albumin (bsa), by Merck Group (59 mentions)
Hydrogen peroxide, by Merck Group (57 mentions)
Ascorbic acid, by Merck Group (55 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (55 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (54 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (51 mentions)
Quercetin, by Merck Group (47 mentions)
Sodium hydroxide (naoh), by Merck Group (45 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (43 mentions)
Methanol, by Merck Group (42 mentions)
Hydrochloric acid (hcl), by Merck Group (39 mentions)
Reduced glutathione, by Merck Group (37 mentions)
Glutathione (gsh), by Merck Group (36 mentions)
Acetic acid, by Merck Group (34 mentions)
Butylated hydroxytoluene, by Merck Group (31 mentions)
Nitroblue tetrazolium, by Merck Group (29 mentions)
Sodium carbonate, by Merck Group (29 mentions)
Ferric chloride, by Merck Group (28 mentions)
649 protocols using thiobarbituric acid
1
Cocaine-Induced Oxidative Stress Evaluation
2
Biochemical Assays for Neurodegenerative Disorders
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Fluorescein sodium salt, 2’,7’-dichlorofluorescin diacetate (DCFDA), Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE), S-butyrylthiocholine chloride (SBTC), Acetylthiocholine iodide (ATCI), Streptozotocin (STZ), glibenclamide (GLB), dithibis-2-nitrobenzoic acid (DTNB), GSH, phenazonium methosulphate (PMS), thiobarbituric acid (TBA), ethylenediamine tetraacetic acid (EDTA), nitro blue tetrazolium (NBT), thiobarbituric acid (TBA), trichloroacetic acid (TCA), hydrogen peroxide (H2O2), triton-X-100, Ferric chloride, hydroxylamine hydrochloride, and bovine serum albumin (BSA) were purchased from Sigma Aldrich, USA and SRL Pvt. Ltd., India. IL-6, TNF-α, hs-CRP, BACE1, BACE2 and Serum insulin (SI) ELISA kits were procured from Wuhan Fine Biological Technology Co., Ltd., China and Monobind Inc., USA respectively. All analytical grade solvents were procured from SRL Pvt. Ltd., Mumbai, India.
3
Oxidative Stress Measurement Assays
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Hydroxylamine·hydrochloride, phenylmethane sulfonylfluoride, thiobarbituric acid (TBA), metaphosphoric acid, o-phthaldialdehyde, bovine serum albumin (BSA), HEPES, digitonin, 2′,7′-dichlorofluorescein diacetate (DCF-DA), tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbo-cyanine iodide (JC-1), polyvinylidene difluoride, iron (III) chloride hexahydrate, protease inhibitor (PI), and polyvinylidene difluoride (PVDF) membrane were purchased from Millipore (Billerica, MA, USA). Superoxide dismutase (SOD) determination kit was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Primary antibody information is presented in Table S1 . Secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
4
Standardized Biochemical Analysis Protocol
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All chemicals used in the present study such as absolute ethanol, chloroform, hydrochloric acid and nitric acid were of high performance liquid chromatography grade. In addition, sodium citrate, 5, 5′-dithiobis-2-nitrobenzoic acid, phenyl methyl sulfonyl fluoride, leupeptin, thiobarbituric acid, and reduced glutathione were from Millipore Sigma, and potassium hydroxide, sodium hydroxide and sodium chloride were from Al-Nasr Pharmaceutical Chemicals Company (Al-Dawa City, ADWIC, Egypt). IQ easy Plus Blood RNA Extraction Kit and Maxime Reverse Transcription Polymerase Chain Reaction (RT-PCR) Premix were from iNtRON Biotechnology (Korea). DNA primer sequences were from Jena BioScience (Germany). PCR Master Mix, magnesium chloride and polymerase chain reaction (PCR) markers were from Promega (USA).
5
Cholesterol and Oxidative Stress Assay
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Questran (Bristol-Myers Squibb, Hounslow, UK) was purchased from a local chemist in Ibadan, Nigeria. Dietary cholesterol and thiobarbituric acid (TBA) were procured from Aldrich Chemical Co. (Milwaukee, WI, USA). Glutathione, hydrogen peroxide, 5,5′-dithio-bis-2-nitrobenzoic acid (DNTB), and epinephrine were purchased from Sigma Chemical Co., Saint Louis, MO, USA. Trichloroacetic acid (TCA) and thiobarbituric acid (TBA) were purchased from British Drug House (BDH) Chemical Ltd., Poole, UK. Other reagents were of analytical grade and the purest quality available.
6
Antioxidant and Enzymatic Assays Protocol
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The following chemicals and compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA): kojic acid, α-amylase solution (ex-porcine pancreas, EC 3.2.1.1), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20), L-glutathione, tyrosinase, 3,4dihydroxy-L-phenylalanine (L-DOPA), acarbos, deoxyadenylic acid oligonucleotide (dA 20 , as a desalted product), concentrated saline sodium phosphate EDTA (20x SSPE; 0.2 mol/L sodium phosphate, 2 mol/L, NaCl, 0.02 mol/L EDTA), phosphate buffer (PBS) pH 7.4, iron (II) sulphate heptahydrate, hydrogen peroxide (30 %, w/v), Folin Ciocalteu reagent, 2,2-diphenyl-1-(2,4,6trinitrophenyl) hydrazyl (DPPH), butylated hydroxytoluene (BHT), trichloroacetic acid (TCA), thiobarbituric acid (TBA), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), paraffin oil and polyphenolic standards -analytical grade and purity ≥ 99% (apigenin-7-О- Germany) . Potassium ferricyanide and ferric chloride were obtained from Zorka (Šabac, Serbia).
Ultrapure water (Thermofisher Scientific, Bremen, Germany) was used to prepare standard solutions and blanks. Syringe filters (13 mm, PTFE membrane 0.45 μm) were purchased from Supelco (Bellefonte, PA, USA). Graphite powder was obtained from Ultracarbon (Dicoex, Spain). All other chemicals and reagents were of analytical reagent grade.
Ultrapure water (Thermofisher Scientific, Bremen, Germany) was used to prepare standard solutions and blanks. Syringe filters (13 mm, PTFE membrane 0.45 μm) were purchased from Supelco (Bellefonte, PA, USA). Graphite powder was obtained from Ultracarbon (Dicoex, Spain). All other chemicals and reagents were of analytical reagent grade.
7
Quantifying Oxidative Stress via TBARS
8
Lipid Peroxidation Quantification Protocol
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Estimation of malondialdehyde (MDA) concentration as an index of lipid peroxidation was assayed according to the method described by Ohkawa et al. [38 (link)], and 1 mL of 20% trichloroacetic acid (Sigma-Aldrich, Inc., Darmstadt, Germany) was added to 1 mL of the tissue homogenate thereafter 2 mL of 0.67% thiobarbituric acid (Sigma-Aldrich, Inc., Darmstadt, Germany) was added. The mixture was incubated at 100 °C for 15 min in a water bath and cooled. Six (6) mL of n-butanol was added and centrifuged at 3000 rpm for 15 min. The absorbance of the clear pink supernatant was then read against a blank at 532 nm spectrophotometrically. The concentration of MDA is expressed in nmol/g of the tissue.
9
Quantifying Lipid Peroxidation in Mouse Paws
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MDA formation was utilized to quantify the lipid peroxidation in the mouse paws and measured as thiobarbituric acid-reactive material. Tissues were homogenized (100 mg/mL) in 1.15% KCl buffer. Two hundred microliters of the homogenates were then added to a reaction mixture consisting of 1.5 mL 0.8% thiobarbituric acid (Sigma, MO, USA), 200 μL 8.1% sodium dodecyl sulfate (Wako, Japan), 1.5 mL 20% acetic acid (pH 3.5), and 600 μL distilled water. The mixture was then heated at 90°C for 45 min. After cooling to room temperature, the samples were cleared by centrifugation (10,000 g for 10 min) and their absorbance measured at 532 nm, using 1,1,3,3-tetramethoxypropane as an external standard [29 (link)]. The level of lipid peroxides was expressed as nmol of MDA/mg of protein.
10
Quantification of Plasma Lipid Peroxidation
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The method developed by Ohkawa et al. [25 (link)] was used in the marking. This method involves the condensation of MDA with thiobarbituric acid, and the formation of a dye compound with 50 μL plasma, 50 μL sodium dodecyl sulphate (SDS; POCH, Gliwice, Poland), 375 mL 20% acetic acid (POCH, Gliwice, Poland), and 375 mL 0.8% thiobarbituric acid (Sigma-Aldrich, Gillingham, UK) was placed in a water bath at 95 °C for 60 min. After incubation, the sample was cooled, and the elution was made into a solution of a dye compound of n-butane (POCH, Gliwice, Poland). After centrifugation, the upper layer of the solution was separated, and the measurement was performed on a multi-detector microplate ELISA reader (Synergy 2 SIAFRT, BioTek, Winooski, VT, USA) at λ = 532 nm. The standard curve was created from a stoichiometrically diluted solution (1,1,3,3-tetramethoxypropane (TMP; Sigma-Aldrich, St. Louis, MI, USA). The concentration of TBARS in the plasma of healthy people was 1–6 μmol/L.
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