A modified technique for the impregnation of lanthanum tracer to study the integrity of tight junctions on cells grown on a permeable substrate

Abstract

Ionic lanthanum is commonly used to trace permeability pathways across epithelia and endothelia in biological electron microscopy. A method for obtaining a uniformly dense precipitate of lanthanum is described. The method, which is a modification of the technique described by Shaklai and Tavassoli (1977) was suitable for fixation of cell cultures grown on permeable filter inserts and was successfully applied to study opening of tight junctions by hypertonic solutions in the airway epithelial cell line 16HBE14o(-). The preparation method formed the basis for a semiquantitative morphological determination in which the tight junctions were subdivided as "intact," "weakened," and "open." By using this modified technique, it could be demonstrated that opening of tight junctions in airway epithelial cells increased, with increasing osmolarity with electrolytes having a stronger effect than nonelectrolytes. A significant linear relationship was found between the osmolarity of the medium and the open state of the tight junctions (as determined by the semiquantitative morphological technique) or the transepithelial electrical resistance.