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DPPH (2,2-diphenyl-l-pycrylhydrazyl), ascorbic acid, Griess reagent, Silymarin (originated from the fruit of Silybum marianum, Cat # S0292), GSH, O-pthaldehyde, 1,1,3,3-tetramethoxypropane, thiobarbituric acid (TBA), tricarboxylic acid (TCA), sodium chloride, nitro blue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH), phenazine methosulphate, sodium pyrophosphate, glacial acetic acid, n-butanol, potassium dihydrogen phosphate, dihydrogen sodium phosphate, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), sodium bicarbonate, thiazolyl blue tetrazolium bromide, trypsin solution, glutamine and JC-1 were obtained from Sigma Co.
The embryos were then blocked (1 hr) in 20% goat serum, incubated overnight in an antidigoxigenin antibody coupled to alkaline phosphatase (1:2,000) (Roche), washed extensively in PBS (at least 6 x 1 hr) and overnight in PBS, followed by a chromogenic nitro blue tetrazolium chloride (NBT) reaction [NBT/BCIP (5-bromo-4chloro-3-indolyl-phosphate, toluidine-salt) tablets, Roche] for 10-30 min, until color was visible.
Acrylamide, NN'-methylenebisacrylamide, sodium dodecyl sulfate, N,N,N',N'-tetramethylethylenediamine, low molecular weight marker proteins, aprotinin, phenylmethylsulfonyl fluoride, [beta]-mercaptoethanol, benzamidine, dithiothreitol, Tris, coomassie blue G, nitro blue tetrazolium, and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Sigma Chemical Co.
The formation of amino blue tetrazolium formazan is in the nicotinamide adenine dinucleotide, phenazine methosulphate and nitroblue tetrazolium (NADH-PMS-NBT), according to the method of Kakkar et al.
The superoxide anions were estimated by the spectrophotometric measurement of the product of the reduction of nitro blue tetrazolium (NBT).