Cortisol Extraction and ELISA Protocol

Larvae were immobilized in ice-cold water and a group of 30 larvae were collected as one sample. After excess water has been removed, the samples were frozen in ethanol (EtOH)/dry-ice bath. Homogenization of the samples was performed with a pellet mixer (VWR International) for 20 seconds. Ethyl acetate was added to homogenate, the supernatant was collected and vaporized. Cortisol was dissolved in 0.2% Bovine serum albumin (A7030, Sigma) in phosphate-buffered saline (PBS) and frozen.
For cortisol ELISA, the minimum cortisol antibody coating time was first determined by the signal-to-noise ratio calculated from wells coated for 1, 3, 6, 16, 24 or 40 hours. For all the other cortisol ELISA experiments, 96-well plates (VWR International) were coated for 16 hours at 4°C with cortisol antibody (P01-92-94M-P, EastCoast Bio) solution (1.6 g/mL in PBS), washed and blocked with 0.1% BSA in PBS. Cortisol samples and cortisol-HRP (P91-92-91H, EastCoast Bio) were incubated at room temperature for 2 hours and washed extensively with PBS containing 0.05% Tween-20 (Roth). Color reactions were performed using Tetramethylbenzidine (TMB:22166-1, Biomol) and Tetrabutylammonium borohydride (TBABH: 230170-10G, Sigma) and stopped using 1M H₂SO₄. For calculating the pH-dependency of the color reaction, diluted HRP with TMB substrate was incubated for 10 minutes at room temperature at different pH levels and read immediately after ending the reaction. Absorbance at 450 nm was read in ELISA plate reader (Multiskan Ascent Microplate Photometer, Thermo Scientific). Comparisons with commercial kits were carried out using Cortisol ELISA Kit (RE52611, IBL International). A detailed step-by-step protocol (Table S1) as well as recipes for all the solutions and stock reagents for the cortisol extraction and ELISA (Table S2) are provided.