Optimized Whole-Mount Fluorescence Staining Protocol for Pulmonary Toxicity Evaluation Using Mouse Respiratory Epithelia

A straightforward whole-mount approach has been developed that uses fluorescence imaging, mouse trachea, and a range of off-the-shelf reagents for rapidly evaluating substance toxicity within the ciliated respiratory epithelium. Using this protocol, the lumen of control trachea samples displays a typical cobblestone epithelial structure, a high density of ciliated cells, and minimal evidence of cell death, as visualized by phalloidin, acetylated tubulin, and fixable live/dead staining, respectively. In contrast, trachea subjected to treatments that induce injury show disrupted epithelial architecture and increased cell death, indicating substance toxicity. These results support the utility of this protocol for rapidly detecting and quantifying respiratory epithelial toxicity and differential cell-type susceptibility.