In the field of protein biology, immunology-based techniques have been evolving for detection and quantification of protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent to other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels, and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., angiopoietin-1 (Ang-1) in the endometrium, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.
Keywords: Adrenal; Endometrium; Estrogen; Immunocytochemistry; Immunofluorescence; Placenta; Proximity ligation assay.