The protocol and study design were approved by the Executive Committee of the Cheyenne River Sioux Tribe Tribal Council (Tribal Resolution number: E-302-08-CR and extended under E-343-2009-CR) and by the University of New Mexico Health Sciences Center Human Research Protection Office (
HRPO number: 08-486).
Hydrogen production assay: Hydrogen peroxide production of neutrophil was assessed based on the horseradish peroxidase (
HRPO) mediated oxidation of phenol red that resulted in formation of a compound demonstrating increase absorbance at 610 nm (Pick and Keisari, 1980).
Labile Po (LPo) was extracted with 0.5 M NaHC[O.sub.3] (pH 8.5), moderately labile Po (MLPo) was extracted with 1M [H.sub.2]S[O.sub.4] and 0.5M NaOH, moderately resistant Po (MRPo) was dissolved in 0.5M NaOH without any precipitation under pH 1.8, and highly resistant Po (
HRPo) was dissolved in 0.5M NaOH with precipitation under pH 1.8:
It uses Hot Rolled Pickled Oiled Coils (
HRPO) bought from other companies to be processed into CRC.
Solution 4- 20 mg horseradish peroxidase (
HRPO; Sigma P-8375) in 0.5 ml deionized and distilled water
The blot was washed thrice in blotting buffer and reacted with horseradish peroxidase (
HRPO) conjugated rabbit anti mouse IgG (Sigma; 1:1000 in blocking buffer) for 1 h.
DTT = dithiothreitol, H&E = haematoxylin and eosin, PAS = periodic acid Schiff, HID = high iron diamine, CsCl = caesium chloride, GuHCl = guanidinium chloride, EDTA = ethylenediaminetetra-acetic acid disodium salt, NEM = N-ethylmaleimide, PMSF = phenylmethysulphonyl fluoride, SDS = sodium dodecyl sulphate, PAGE = polyacrylamide gel electrophoresis, NaCl = sodium chloride,
HRPO = horseradish peroxidase, TBST = tris-buffered saline tween-20, TBS = tris-buffed saline tween, [V.sub.i] = included volume, [V.sub.o] = void volume.
The macroarrays were subjected to stringent wash conditions (three 30-min washes with 2x SSC containing 1 g/L SDS at 50[degrees]C; one 30-min wash with 0.2x SSC containing 1 g/ L SDS at 45[degrees]C; and one 1-h wash in 2x SSC at room temperature) and developed with DNA Detector
HRPO reagents (KPL) essentially according to the manufacturer's instructions.
The assay was based on horseradish peroxidase (
HRPO)mediated oxidation of phenol red by [H.sub.2][O.sub.2], leading to the formation of a compound that absorbs at 610 nm.
Posteriormente se agregaron 50 [micron]l de solucion conjugado de antigeno p26 con peroxidasa de rabano picante (
HRPO), se mezclaron y se incubaron por 30 minutos a 37[grados]C, tras tres lavados para eliminar los restos de solucion se colocaron 100 pl de solucion sustrato, se incubaron por 15 minutos a temperatura ambiente.