Plasma estradiol from mouse peripheral blood was quantified using estradiol assay kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Briefly, murine peripheral blood was collected from tail vein in the presence of heparin and centrifuged at 1000g for 10 min at 4°C to separate the cell and plasma fractions. Samples were pretreated with pretreatment E solution and centrifuged, and supernatant was mixed with pretreatment F solution. To wells coated with estradiol antibody, samples and estradiol conjugate were added and incubated for 2 hours at RT on a shaker. Wells were washed, and substrate solution was added for 30 min at RT and protected from light. Then, stop solution was added, and measurements were performed with a plate reader (Tecan Infinite 200 PRO) using Ex450 and Em540 nm filters.

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