2.8 Immunohistochemical assays
Immunohistochemistry analysis was performed by using a DAB staining kit (Gene Tech Co. Ltd., Shanghai, China; GK347011) to determine the expression levels of StAR (1:1000 dilution) in adrenals at GD20, PW12, and PW28. Adrenals were cut into 5 μm-thick slices along the longitudinal axis. For immunohistochemical staining, the slices were dewaxed and washed in PBS. After antigen retrieval, adrenal sections were blocked in 5% blocking serum at room temperature for 30 min and probed with primary antibodies overnight at 4℃. After primary antibody incubation, sections were incubated with a biotinylated secondary antibody and then with an avidin-biotinylated horseradish peroxidase complex solution according to the manufacturer’s directions. Finally, peroxidase activity was determined with a DAB staining kit (Gene Tech Company, Ltd., Shanghai, China). All the images were analyzed using Image-Pro Plus software (version 6.1, Media Cybernetics, Silver Spring, USA). The intensity of StAR staining was determined by measuring the mean optical density in five random fields for each sample, and five sections from different samples per group were used for quantification.