Quantification of horizontal and vertical distribution of junctional proteins in fixed epithelial cells

Polarized epithelial cells form a tightly packed monolayer where individual cells are connected by cell-cell junctions, including tight junctions (TJ) and adherens junctions (AJ). Here, we present techniques for quantifying the horizontal and vertical distribution of junctional proteins in confluent, fixed epithelial cells. This approach is utilized to evaluate variations in the intensity and localization of the proteins that compose the AJ and TJ under different experimental conditions. Although our protocol is optimized for Madin-Darby Canine Kidney (MDCK) cells, it is adaptable to any cell line capable of forming cell-cell junctions. For complete details on the use and execution of this protocol, please refer to Rabino et al. (2024). ¹.