Eia buffer

Because the number of pretest plasma samples was small in each group, the plasma cortisol concentrations of fish that did not undergo the mirror-image test (pretest) were combined and evaluated. Five microliters of plasma was extracted using 2 mL of diethyl ether and resuspended in 500 μL of enzyme immunoassay (EIA) buffer (Cayman Chemical, Ann Arbor, MI, USA). The concentrations of the hormones were measured using a commercially available EIA kit (Cayman Chemical) following the manufacturer’s instructions. All samples were measured in triplicate. The cross-reactivity of antibodies is shown in Table 3. The inter- and intra-assay coefficients of variation were 14.7 and 7.3% for E₂, 8.2 and 8.3% for 11-KT, and 7.6 and 8.4% for cortisol, respectively.
Table 3

Cross-reactivity of antibodies used for enzyme immune assay of plasma estradiol (E₂), 11-ketotestosterone (2 11-KT), and cortisol

estradiol		11-ketotestosterone		cortisol
Compound	Cross reactivity		Cross reactivity		Cross reactivity
estradiol	100%	11-ketotestosterone	100%	cortisol	100%
estorone	12%	adrenosterone	2.9%	corticosoterone	0.14%
estriol	0.30%	4-androstan-11β, 17β-diol- 3-one	0.01%	cortisone	0.13%
androstendiol	0.02%	5-androstan-17β-ol- 3-one	<0.01 %	androstendione	<0.01 %
testosterone	<0.01 %	testosterone	<0.01 %	testosterone	<0.01 %

A Competitive Enzyme Immunoassay (DRG International, Inc., Springfield, MO, USA) was performed on the serum of both SC and IC groups in accordance with the manufacturer’s recommendations. Briefly, 96-well plates precoated with pre-blocked anti-rabbit monoclonal mouse IgG were incubated with experimental animal serum (1:50) in conjunction with Testosterone-bound Acetylcholinesterase and rabbit antiserum testosterone in EIA buffer (Cayman Chemical Company, Ann Arbor, MI, USA) for two hours at room temperature. Results were read at 405 nm after a one-hour incubation with Ellman’s Reagent (Cayman Chemical Company), according to the manufacturer’s recommendations (catalog EIA-155996).

Steroid extraction from plasma and measurement of P4, T, and E2 levels were performed following the methods described previously [4 (link), 30 (link)]. Briefly, 400 µl of diethyl ether was added to 100 µl plasma in a glass test tube, vortexed, and allowed to separate. The upper diethyl ether layer was recovered, and this extraction procedure was repeated three more times. The diethyl ether from repeated extractions was pooled and dried under a nitrogen stream at 37ºC. Samples were reconstituted with 100 µl of EIA buffer (Cayman Chemical Company, Ann Arbor, MI, USA) and stored at –30 ºC until measurement.
Plasma concentrations of E2, T, and P4 were measured using commercially available ELISA kits (Cayman Chemical Company) following the manufacturer’s instructions [4 (link)]. In an ELISA plate pre-coated with secondary antibody, 50 µl of extracted plasma sample was mixed with 50 µl of AChE tracer and 50 µl of primary antibody. The assay plate was then sealed and incubated at 4ºC overnight. After washing five times with wash buffer, 200 µl of Ellman’s reagent was added to each well for color development at 25ºC for 60 to 100 min. For the rapid protocol to monitor the daily P4 levels, the incubation with primary antibody was conducted at 25ºC for 60 min. Absorbance of each well was measured at 405 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific).

20E levels were quantified using the 20E EIA kit (Cayman Chemical). Nine females per time point were collected into the microtubes, snap frozen and kept at −80 °C. On the day of analysis, all samples were homogenized in 500 µl methanol using steal beads and a Qiagen tissue lyser at 50 rpm for 10 min. Samples were centrifuged at 15,000 rpm for 1 min and the supernatant (420 µl) was transferred to a clean tube. The methanol was evaporated in a speedvac. The pellets were dissolved in 230 µl of EIA buffer (Cayman Chemical). A 1:3 dilution series of 20E starting from 361 ng/ml served as a standard curve. Measurements were performed according to the manufacturer’s instructions.

Flies were reared at 25°C and aged for 4 days. Ovaries were dissected in PBS. Samples were homogenized in 50 μL of methanol in 1.5-mL tubes using a pestle. After centrifugation at 20,913 x g for 1 min, the supernatants were transferred to new tubes and dried with a centrifugal evaporator. Samples were resuspended in 50 μL of EIA buffer (Cayman Chemicals) according to the manufacturer’s protocol and incubated at 4°C overnight. Ecdysteroid levels were quantified by an enzyme-linked immunosorbent assay using anti-20E antiserum (Cayman Chemicals) and 20E-acetylcholinesterase (Cayman Chemicals) as essentially described [62 (link)]. Note that the antiserum used in this study is known to recognize not only 20-hydroxyecdysone (20E) but also ecdysone [63 (link)]. In this study, we used we used 20E (ENZO Life Sciences) as a standard, and the ecdysteroid amount was expressed in 20E equivalents. Absorbance was measured at 415 nm using a microplate reader Multiskan GO (Thermo Fisher Scientific).