The immunohistochemical assay was carried out using the method described in previous studies.27 Briefly, the brain samples post-fixed with 4% PFA were coronally cut into 5 μm hypothalamus sections, which contain paraventricular nucleus. The brain sections were dewaxed and incubated in 3% hydrogen peroxide for 10 min, and then boiled in citrate buffer (pH 6.0) for 5 min. After blocking with goat serum for 30 min, the sections were incubated with anti-CRH antibody (dilution 1: 100, Abcam, Britain) and anti-glucocorticoid receptor (GR) antibody (dilution 1: 100, Bioss, China) overnight at 4°C. Subsequently, the secondary antibody was incubated at 37°C for 30 min, and specific labeling was visualized using a 3,3ʹ-diaminobenzidine (DAB) kit. Images were captured via a digital microscope (BA200 Digital, McAudi) and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA). The CRH and GR expression in the hypothalamus were measured by the average optical density (AOD) to compare the potential differences between groups.

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