Ka1883

Aldosterone serum levels were measured in mice using a commercial kit (KA1883; Abnova, Walnut, CA, USA) and the assay was performed according to manufacturer's instructions. The assay was performed on blood serum isolated from aldosterone-treated (4 weeks) mice. Blood (300 μl) was collected by puncturing the heart. Then, blood samples were centrifuged at 1,500 r.p.m. for 15 min at room temperature and the serum was transferred to a 1.5 ml tube. Blood serum isolated from vehicle-treated mice was used as control.

Aldosterone concentration in serum was measured using a competitive ELISA (Abnova, KA1883) according to the manufacturer's protocol. 50 μl of standards, control and serum samples from adult MSUS and control males was added to wells containing aldosterone antibody, and an HRP‐conjugated aldosterone antigen was added. After 1‐h incubation at room temperature for competitive binding, cells were washed and tetramethylbenzidine (TMB) substrate was added for colour development, followed by addition of stop solution. Absorbance was measured at 450 nm with a NOVOstar Plate Reader (BMG Labtech), and sample absorbance (inversely related to concentration) was determined by plotting a standard curve with manufacturer‐supplied standards and controls. Samples were measured in duplicate and averaged.