Colon tissue samples were extracted by mixing with methanol and then washed with hexanes as previously stated [52 (link)]. Briefly, the extractions were diluted with PBS for the ELISA analysis. The ricinoleic acid (NuChek Prep Inc. Elysian, MN, USA)–ovalbumin (Sigma-Aldrich) antigen (1 μg/mL) was coated on the plate at 4 °C overnight. After blocking with 3% skim milk, 50 μL of the samples (50 times dilution) or DiHOMEs standard solution was equally mixed with 1 μg/mL polyclonal antibody for 1 h at room temperature. After washing, Goat-anti-Rabbit IgG-HRP (Fitzgerald Industries International, Concord, MA, USA) was added for another 1 h. Finally, the 3,3′5,5′-tetramethylbenzidine (Sigma-Aldrich) detection reagent was used for color development and 1 M H2SO4 (Sigma-Aldrich) was applied to stop the reaction. The absorbance was measured at 450 nm and the final concentrations of DiHOMEs were calculated using the standard curve.
1 m h2so4
Manufactured by Merck Group
Sourced in United States
1 M H2SO4 is a concentrated sulfuric acid solution with a molarity of 1 mole per liter. It is a commonly used reagent in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Avidin biotin complex solution, by Vector Laboratories (1 mentions)
Biotinylated anti mouse antibody, by Vector Laboratories (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
Gc 17a, by Shimadzu (1 mentions)
Methanol, by Merck Group (1 mentions)
3 protocols using 1 m h2so4
1
DiHOMEs Quantification by ELISA
2
Quantifying Neuronal Differentiation in N2A Cells
3
Membrane Fatty Acid Profiling Protocol
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The methods used for membrane fatty acid analysis have been described previously [16] [17] [18] . In brief, 100-150 mg of tissue was weighed and homogenised in a chloroform-methanol mixture (2:1, v/v) (analytical grade, Thermo-Fisher Scientific, North Ryde, Australia; Sigma-Aldrich, Castle Hill, Australia). Steps were taken for acidified total lipid extraction using 1 M -H 2 SO 4 (Sigma-Aldrich), solid phase phospholipid separation via silica Sep-Pak® columns (Waters, Sutton, MA, USA), transesterification of phospholipid fatty acids using 14% boron trifluoride in methanol ([Sigma-Aldrich] stored at 0-4°C) heated at 85°C for 1 h, purification via Sep-Pak® Florisil columns (Waters) using diethyl ether (5%) (Fluka; Sigma-Aldrich) in petroleum spirit (7 mL) (Fluka; Sigma-Aldrich) and finally GC (Shimadzu GC-17A, 30 m × 0•25 mm internal diameter capillary column, total run time 23 min; Shimadzu, Rydalmere, Australia). All solvents were freshly prepared at the time of analysis and contained 0•01% (w/v) butylated hydroxytoluene (Sigma-Aldrich). Individual fatty acids were identified by comparison with the known standards in the laboratory. The relative amount of each fatty acid was determined by integrating the area under the peak and dividing by the result for all fatty acids detected.
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