Paw tissues of animals submitted to paw edema test were collected 6 hours after carrageenan administration. Samples were weighted, ground, and kept in phosphate buffer [50 mM, pH 6.0, and 0.5% hexadecyltrimethylammonium bromide (HTAB, Sigma Chem. Co, USA)]. Samples were centrifuged at 12.000 ×g for 2 minutes at 4°C. Each well in a 96-well plate was filled with 5 μL of centrifuged supernatant and 200 μL of o-dianisidine dihydrochloride solution (Sigma Chem. Co, USA; 0,167 mg/mL, prepared in 50 mM potassium phosphate buffer with 0.005% H2O2) following spectrophotometer reading (450 nm). Results were expressed in MPO units (UMPO/mg of tissue), considering that 1 UMPO corresponds to the quantity that one enzyme degrades with 1 μmoL per minute [19 (link)]. The test was performed in duplicates.
O dianisidine dihydrochloride solution
Manufactured by Merck Group
O-dianisidine dihydrochloride solution is a chemical reagent commonly used in analytical and biochemical applications. It serves as a substrate for various enzymatic reactions, particularly in the detection and quantification of specific analytes. The solution provides a consistent concentration and formulation of the o-dianisidine dihydrochloride compound, facilitating reliable and reproducible experimental results.
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Lab products found in correlation
2 protocols using o dianisidine dihydrochloride solution
1
Myeloperoxidase Activity Assay in Paw Edema
2
Measuring Ceruloplasmin Levels in Mice
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Ceruloplasmin levels were measured weeks 1, 2, 4, and 6 during the study period. Nontumor bearing mice were treated with ATN-224 (0.7 mg/kg) or PBS by daily gavage and blood was collected from facial vein every weeks. Ceruloplasmin was assayed based on its oxidase activity. Two tubes each containing 25 μl mouse serum and 375 μl of 0.1 mol/l sodium acetate buffer (pH 5.0) were incubated for 5 minutes in a 30 °C water bath. Prewarmed 100 μl o-dianisidine dihydrochloride solution (7.88 mmol/l; Sigma) was then added to each tube and the reaction was allowed to continue for 5 minutes in one tube and 15 minutes in the other. Finally, the reaction was quenched by adding 1 ml of 9 mol/l sulphuric acid. The absorbance of both tubes was measured at 540 nm using Beckman Coulter DU 530 spectrophotometer. Ceruloplasmin concentration in IU was calculated using the formula ceruloplasmin oxidase activity = (A15–A5) × 0.625 units/ml, where A5 and A15 are the absorbance at 5 and 15 minutes, respectively.
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