Serum Corticosterone Quantification Protocol

Serum samples were collected, and corticosterone quantification was performed, as described before [82 (link)]. In brief, serum was mixed with a dexamethasone (Cayman Chemicals, Ann Arbor, MI, USA) standard and a fixed amount of corticosterone (Sigma Aldrich, St. Louis, MO, USA) to monitor and facilitate measurement of the endogenous levels. Samples and standards were extracted two times and subjected to reversed-phase HPLC analysis. The HPLC (automatic liquid autosampler and isocratic pump: Series 1100; thermostatted column compartment and variable wavelength detector: Series 1200, all Agilent Technologies, Waldbronn, Germany) was equipped with an ODS Hypersil C18 column (5 µm, 150 mm × 4 mm; MZ-Analysentechnik, Mainz, Germany) and temperature set at 24 °C. Detection was carried out at 245 nm with a consistent flow rate (1.2 mL/min). Chromatograms were obtained and analyzed by ChemStation for LC software (Rev. A.10.02, Agilent Technologies, Waldbronn, Germany).