Canonical stress signaling pathways involve adaptive neuroendocrine responses. Typically, stress activation of the hypothalamic–pituitary–adrenal axis will result in an increase in glucocorticoids that act on almost all tissues including the brain. Recent work has investigated the impact of glucocorticoid signaling in astrocytes, highlighting sensitivity of these cells to stress hormones. Further understanding whether specific glucocorticoid signaling changes exist in astrocytes and whether regional heterogeneity exists remains unknown. Here, we outline a method to investigate glucocorticoid signaling in astrocytes in fixed tissue.