Solution Edos and you may Ad was in fact counted using Estradiol EIA Package (Cayman Toxins, MI) and androstenedione chemical immunoassay system (Oxford Biomedical Look Inc., MI), respectively. ELISAs off T and you may E1 had been performed depending on the same procedure for 17,20?-P (Matsuyama et al., 1998b) and discussed less than briefly.
Stock solutions: Coating buffer, 0.05 M carbonate buffer pH 8.4, containing 0.05% NaNstep 3; washing solution, 0.85% NaCl; blocking solution, 0.05 M PBS containing 0.1% BSA, 3% sucrose, and 0.005% thimerosal; assay buffer, 0.05 M borate buffer, pH 7.8, containing 0.1% BSA and 0.01% thimerosal; substrate solution, 0.2 M citrate buffer, pH 4.5, containing 0.01% H2O2, added 0.5% o-phenylenediamine immediately before use; stopping solution, 6 N H2SO4. All the solutions except for the stopping solution were stored at 4°C.
Second Antibody-coated Microtiter Dishes: Microtiter plates (MS-3596 F/H plate, Sumitomo Bakelite Co.) was indeed coated having 100 ?l for every single better regarding goat anti-bunny IgG (15 ?g/ml within the level boundary). The latest plate was close and incubated at the 4°C to possess 48 hours. Shortly after elimination of unbound antibody, the newest wells have been clean 3 times with laundry solution and dried. Clogging provider (two hundred ?l) are put into for every well, that was upcoming closed and incubated during the 4°C to have 24 hours.
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