Protocol for quantifying the 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 adherens junctions (AJs) and tight junctions (TJs). Here, we present a protocol for quantifying the horizontal and vertical distribution of junctional proteins in confluent, fixed epithelial cells between different experimental conditions. We describe steps for sample preparation, immunofluorescence, and image acquisition. We also provide detailed instructions on how to use Fiji/ImageJ and RStudio for this procedure without any prior experience necessary. For complete details on the use and execution of this protocol, please refer to Rabino et al.¹.