2030 multilabel reader arvo x4

Plasma LH concentrations were measured using a time-resolved fluorometric immunoassay (DELFIA System; PerkinElmer, Branchburg, NJ, USA). Briefly, purified rat
LH (LH-I-9, NIDDK) was labeled with europium (Eu) using a DELFIA Eu-Labeling Kit (PerkinElmer). Anti-rabbit gamma-globulin goat serum was prepared in our
laboratory and purified by ammonium sulfate precipitation. A 96-well immunoplate (Nunc, Tokyo, Japan) was coated with anti-rabbit gamma globulin and then
overlaid with NIDDK anti-rat LH-S-11 (1:4,000 dilution). Diluted plasma samples and LH standards (NIDDK rat LH-RP-3) were incubated at 4°C overnight, and
optimally diluted Eu-labeled hormone was added for 2 h at 20°C. After washing, the plates were treated with DELFIA Enhancement Solution (Perkin Elmer) and
specific fluorescence (excitation wavelength 340 nm and emission wavelength 613 nm) was measured on a Multilabel Reader 2030 ARVO X4 (Perkin Elmer). The intra-
and inter-assay coefficients of variation for LH were 6.5% and 9.9%, respectively.

NADPH oxidase activity was measured using a lucigenin assay. Mouse liver tissues were homogenized in 50 mM phosphate buffer (pH 7.0) containing 150 mM sucrose, 1 mM EDTA, and a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The reaction was initiated by adding the homogenates (10 μg of protein) to an assay solution containing lucigenin (5 μM) and NADPH (100 μM), and photon emissions were measured for 3 min in a luminometer chamber (Multilabel Reader 2030 ARVO X4; PerkinElmer, Waltham, MA).
Liver tissue O₂⁻ generation was detected by Dihydroethidium (DHE, Beyotime, China) following the specification guide book.

Plasma luteinizing hormone (LH) concentrations were measured using a time-resolved fluorometric immunoassay (DELFIA System; PerkinElmer, Branchburg, NJ, USA). Purified rat LH (LH-I-9,
National Institute of Diabetes and Digestive and Kidney Diseases, NIDDK, Torrance, CA, USA) was labeled with europium (Eu) using a DELFIA Eu-Labeling Kit (PerkinElmer, Waltham. MA, USA).
Anti-rabbit gamma globulin goat serum was prepared in our laboratory and purified via ammonium sulfate precipitation.
A 96-well immunoplate (Nunc, Tokyo, Japan) was coated with anti-rabbit gamma globulin and overlaid with a 1:4,000 dilution of NIDDK anti-rat LH-S-11 (NIDDK). Diluted plasma samples and LH
standards (rat LH-RP-3, NIDDK) were incubated at 4°C overnight, and optimally diluted Eu-labeled hormones were added for 2 h at 20–25°C. After washing, the plates were treated with DELFIA
Enhancement Solution (PerkinElmer), and specific fluorescence (excitation and emission wavelengths of 340 and 613 nm, respectively) was measured using a Multilabel Reader 2030 ARVO X4
(PerkinElmer). The intra- and inter-assay coefficients of variation for the LH levels were 6.5% and 9.9%, respectively.