Cortisol elisa kit

Cortisol concentration of control larvae and three exposure concentrations (0.2, 2, 20 mg L⁻¹) were measured after 2 days of exposure at 120 hpf according to a protocol previously described by Tudorache et al.^{21 (link)}. Briefly, 15 larvae per replicate were pooled and 6 replicates per group sampled. The larvae were snap-frozen in liquid nitrogen and then homogenised in 500 µL of phosphate-buffered saline (PBS) using a bullet blender. Cortisol was extracted with two volumes of diethyl ether three times. After evaporation of diethyl ether overnight, the cortisol was redissolved in 0.2% bovine serum albumin (BSA) in PBS. Larval cortisol measurements were carried out using a cortisol ELISA kit (Abnova KA1885) according to the manufacturer’s instructions and absorbance read at 450 nm using a plate reader (Tecan Infinite M1000 PRO).

A commercial ELISA kit (Cortisol ELISA Kit, Abnova, Taipei, Taiwan) was used to analyze plasma cortisol concentrations according to the manufacturer’s instructions. The procedure involved several steps: (i) 25 µL of goat plasma samples was added to coat the 96-well microplates, (ii) 100 µL of cortisol enzyme conjugate solution was added, (iii) incubation at 37 °C, (iv) the plates were washed four times, (v) 100 µL of color reagent (3,3′,5,5′ -tetramethylbenzidine, TMB) was added to each well, and then (vi) 50 µL of the stop solution was added to stop the reaction. A Synergy HTX Microplate Reader (Bio-Tek, Winooski, VT, USA) was used to measure the absorbance at 450 nm, and finally, the cortisol concentrations were determined against a standard curve created using the standards provided by the manufacturer.

A commercially available kit (Cortisol ELISA Kit, Abnova, Taipei, Taiwan) was used to analyze plasma cortisol concentrations following the instructions provided by the manufacturer. Briefly, microplates (96-wells) were coated with 25 μL of goat plasma samples followed by the addition of 100 μL of cortisol enzyme conjugate solution to each sample. After 1 h of incubation at 37 °C, the plates were washed four times, 100 μL of color reagent (3,3′, 5,5′ -tetramethylbenzidine, TMB) added to each well, and then 50 μL of the stop solution was added to stop the reaction. Absorbances were measured at 450 nm using a Synergy HTX Microplate Reader (Bio-Tek, Winooski, VT). The cortisol concentrations were determined against a standard curve. This sheep cortisol kit has been validated for goats in our laboratory. The minimum detectable concentration of cortisol by this method is 1.0 ng/mL.