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Methoxychlor-Induced Atresia: MATERIALS AND METHODS(8)

METHODS(8)

Follicle Stimulating Hormone and Luteinizing Hormone Assays

Blood was collected from CD-1 mice during estrus and FSH and LH assays were carried out by radioimmunoassay (RIA) using reagents from the National Hormone and Pituitary Distribution Program. For FSH, we conducted measurements at all doses (8-64 mg/kg MXC); however, insufficient blood remained to measure LH at all doses, so we chose to measure the control and 32 mg/kg dose, a dose at which we observed an effect of MXC on the ovary. Rat FSH and LH hormone antigen, rat FSH and LH antiserum, and mouse FSH and LH reference preparation were provided by the National Institute of Diabetes and Digestive and Kidney Diseases.

Iodination reagents (IODO-BEADS 28665, 28666) were purchased from Pierce (Rockford, IL). For both FSH and LH, a standard curve was prepared and cold standards and samples (100 |xl) were added to labeled tubes along with primary antibody (FSH at 1:1400 dilution and LH at 1:500) and iodinated FSH or LH. Samples were shaken and stored at 4°C overnight. On Day 2, secondary antibody was added (1:10 dilution) along with 2% normal rabbit serum (Sigma Aldrich, St. Louis, MO) and incubated at room temperature for 5 min. The tubes were centrifuged for 15 min at 3000 rpm, supernatant was decanted, and pellets were counted in a gamma counter for 1 min each.