Microvascular endothelial expression of estrogen receptors. Estrogen receptor-β (red, ER-β, Thermo Fisher, MA5-13304; green, CD31, R&D systems, AF3628; blue, DAPI) expression in untreated arterioles from females <40 yr (n = 3; A) and ≥40 yr (n = 4; B), and biological males (n = 3; C). D: mean intensity of ER-β normalized to endothelial area identified using CD31 staining. G protein-coupled estrogen receptor (red; Thermo Fisher, PA5-77396) costained with CD31 (green; R&D systems, AF3628) and DAPI (blue) in arterioles from females <40 (n = 3; E) and ≥40 (n = 4; F) yr of age, as well as biological males (n = 3; G). H: mean intensity of GPER-signal normalized to endothelial area identified using CD31 staining. Top, right: for visualization purposes only, magnified sections of each arteriole with a 40% increase in both the brightness and contrast. Representative immunohistochemical staining for estrogen receptor-α (Thermo Fisher, MA5-14501) in females <40 (I) and ≥40 (J) yr of age, as well as biological males (K). *P < 0.05: one-way ANOVA with Fisher’s least significant difference (LSD) test (D and H). GPER, G protein-coupled estrogen receptor; ER-β, estrogen receptor-β.