Epoch microplate spectrophotometer plate reader

E₂ levels were measured in the DH using an EIA assay following dual liquid-solid phase extraction (Chao et al., 2011 (link)). Briefly, frozen DH tissue was first homogenized in 0.1 M phosphate buffer, followed by three rounds of ether extraction. The final organic phase was dried under air in a 50 C water bath, followed by re-suspension in 250 μl of 0.1 M phosphate buffer prior to solid phase extraction. Re-suspended samples were then eluted through C18 columns under vacuum pressure and washed with ddH₂O to elute hydrophilic polar compounds. Hydrophobic compounds, including steroids, were then eluted with a series of washes with 100% methanol, followed by an evaporation step under air in a 50 C water bath. After drying, samples were suspended in EIA buffer. E₂ concentrations were then measured from EIA plates per the manufacturer’s instructions using an Epoch Microplate Spectrophotometer plate reader (Biotek) with a 450 nm filter and Gen5 Software (Chao et al., 2011 (link)). The EIA assay used to measure brain estrogen levels in this experiment is highly specific for E₂ (cross-reactivity: 14% for E₂-3-glucuronide; 12% for estrone; 1% for E₂-17-glucuronide; < 0.10% for other major steroids including testosterone; Cayman chemical). Data are expressed in pg E₂ per mg wet weight of DH tissue, after correcting for recovery (recovery was 60.6% and intrassay CV was 2.17%).