Estradiol Quantification in Zebra Finch Brain

Following steroid extraction using a combined liquid and solid-phase extraction protocol, samples were resuspended in 300 μl of EIA buffer (Cayman Chemical, Ann Arbor, MI) and vortexed on low speed for 1 minute. Samples were assayed in triplicates using an E₂ Enzyme Immunoassay kit (Cayman Chemical) previously validated for use with zebra finch brain tissue according to manufacturer's instructions (30 (link)–32 ). Prior to ether and solid-phase extraction, an additional sample was spiked with radioinert E₂ to the concentration of 256 pg/mL, processed in an identical manner, and assayed in triplicates alongside the experimental samples in order to estimate recovery. To ensure a lack of interference from our tissue matrix and reliability of measurement, we ran two-fold dilutions and validated recovery of the corrected concentration compared to the identical undiluted sample.

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Mehos C.J., Nelson L.H, & Saldanha C.J. (2016). A quantification of the injury-induced changes in central aromatase, estrogenic milieu and steroid receptor expression in the zebra finch. Journal of neuroendocrinology, 28(2), 12348.

Publication 2015

EIA buffer

Brain Buffer Cayman Dilutions Enzyme immunoassay Ether Finch Solid phase extraction Steroid Tissue Zebra

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Variable analysis

independent variables

Steroid extraction protocol (combined liquid and solid-phase extraction)

dependent variables

Estradiol (E2) concentration

control variables

Brain tissue of zebra finch
E2 Enzyme Immunoassay kit (Cayman Chemical) previously validated for use with zebra finch brain tissue
Resuspension in 300 μl of EIA buffer (Cayman Chemical, Ann Arbor, MI) and vortexing on low speed for 1 minute
Assay in triplicates

positive control

An additional sample spiked with radioinert E2 to the concentration of 256 pg/mL, processed in an identical manner, and assayed in triplicates alongside the experimental samples to estimate recovery

negative control

Two-fold dilutions of samples to validate recovery of the corrected concentration compared to the identical undiluted sample, ensuring a lack of interference from the tissue matrix and reliability of measurement

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