The Sandwich Assay: A Method for Subcellular Visualization of Paracellular Macromolecule Passage in Epithelial Sheets

To date, the permeability of epithelia to larger solutes (greater than ∼4 Å in diameter) has been analyzed by flux measurements using various tracers that cannot spatially resolve the permeation sites. This unit describes a method for localizing such sites of passage in epithelial sheets with subcellular resolution. The method makes use of avidin as a basolateral capture probe in epithelial monolayers or mucosae to unmask the passage of biotinylated and fluorophore-labeled tracer molecules as they go through the junctional barrier. Once bound to avidin, the tracers are immobilized at the site of a barrier leak. The localization, the distribution, and the extent of passage are eventually evaluated by imaging. The assay detects single leaks and is hence able to spatially resolve rarely occurring changes. It is also modular and flexible to use with various macromolecular tracers, and its sensitivity is adjustable. If designed as a chase experiment, the method allows for analysis of temporal barrier openings. If performed at low temperatures, this assay will block transcellular passage and, combined with global flux measurement, unambiguously determine paracellular passage. © 2018 by John Wiley & Sons, Inc.