A Double-Label Immunohistochemical Method for Assessment of Epithelial Cell Replication in Paraffin Sections of Toxicant-Damaged Lungs

Abstract

Enhanced epithelial cell replication is a sensitive indicator of toxicant-induced lung damage. Cell identification in the deep lung can be difficult and limit the usefulness of conventional techniques for examining cell-cycle kinetics in toxicant-damaged lungs. The purpose of this study was to develop a technique to reliably differentiate epithelial from nonepithelial cells in the deep lung. Formalin fixed, paraffin sections of toxicant-exposed lungs were used to develop a double, sequential, peroxidase/alkaline phosphatase method to detect bromodeoxyuridine and cytokewtins. Use of lü-5 cytokeratin antibody, which binds to all cytokeratin polypeptides and is epithelial cell specific, and a biotinstreptavidin amplified detection system maximized the detection of cytokeratin immunoreactivity and allowed cells with a negative reaction to be classified as nonepithelial. The staining produced was distinct and readily detected by light microscopy. In foci of severe damage, the cytokeratin immunostaining of epithelial cells highlighted the general architecture of the parenchyma. Cell counts and labeling data from inflammatory foci in endotoxin-instilled rat lungs were compared using the double-label versus a conventional single-label immunohistochemical method. The proportion of cells that could be identified was significantly increased in the double-label slides. We conclude that in lungs with severe damage, where the usual morphologic features such as cell size, shape, vacuolization, and position are unreliable indicators of cell type, the double-label procedure would aid the microscopist in obtaining reliable cells counts. (The J Histotechnol 25:15, 2002)Submitted: September 10, 2001; Accepted with revisions: November 7, 2001