Tissue sections from the proximal and distal parts of the small and large intestines were collected after being flushed with PBS to clean from their contents and snap-frozen in isopentane on dry ice and then stored at −80 °C until measurements. Samples were weighed, homogenized in 0.02 M phosphate buffer (PB), pH 7.4, and centrifuged at 13,500× g for 5 min. The pellet was resuspended in 0.05 M PB, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide and frozen at −20 °C for 2 h, then thawed at 25 °C, followed by sonication. Thereafter, the samples were placed in a water bath at 60 °C for 2 h and then centrifuged again. The supernatants were used, and a tetramethylbenzidine liquid substrate system (Sigma-Aldrich) was added as a peroxidase substrate, and the mixture was incubated in the dark for 10 min at room temperature. To stop the reaction, 0.5 M H2SO4 was used. The enzyme activity was determined spectrophotometrically, as the MPO catalyzed change in absorbance at 450 nm in the redox reaction of H2O2 at 25 °C. Values are expressed as MPO units per gram of tissue.
Tetramethylbenzidine liquid substrate system
Manufactured by Merck Group
Sourced in Denmark
The Tetramethylbenzidine liquid substrate system is a colorimetric reagent used in various analytical and diagnostic applications. It serves as a substrate for enzyme-based detection assays, generating a colored product upon enzymatic conversion. The core function of this product is to provide a visible signal in these types of assays.
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Lab products found in correlation
2 protocols using tetramethylbenzidine liquid substrate system
1
Myeloperoxidase Activity Quantification in Intestinal Tissue
2
Quantifying Circulating FHR-5 Levels
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Circulating FHR-5 was detected by enzyme-linked immunosorbent assay as previously described. 17 Briefly, rabbit anti-human FHR-5 antibody (Abnova Corporation, Taipei, Taiwan) was coated onto half the wells of a microtitre plate (Nunc Immunoplate, Roskilde, Denmark). After blocking with 1% bovine serum albumin (Sigma Chemical Company, St. Louis, MO), the plasma samples were added and incubated for 1 hour at room temperature. Binding of FHR-5 was examined using mouse anti-human FHR-5 antibodies (Abnova Corporation). Following the addition of horseradish peroxidaseconjugated rabbit anti-mouse IgG (DakoCytomation, Glostrup, Denmark), the reaction was developed using the peroxidase chromogenic substrate 3,3 0 ,5,5 0 -Tetramethylbenzidine Liquid Substrate System (Sigma Chemical Company) and stopped with 1 mol/l sulfuric acid before the absorbance was measured at 450 and 570 nm. Serial dilutions of recombinant human FHR-5 protein (R&D Systems, Minneapolis, MN) were used to establish the standard curve, which was then used to calculate circulating FHR-5 levels.
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