Newly developed technique for dual localization of keratins 13 and 14 by fluorescence immunohistochemistry

It is difficult to visualize histological details on semi-ultrathin sections by light microscopy after immunohistochemical labeling because the histological structures in such sections cannot be distinguished by standard counterstaining. To solve this problem and to visualize the immunoreactivity of keratins 13 (K13) and 14 (K14), we used a newly developed technique for dual localization of antigens by fluorescence immunohistochemistry and confocal laser-scanning microscopy in transmission mode, after staining specimens with toluidine blue. Using this approach, we examined the immunolocalization of K13 and K14 on the lingual epithelium of fetal and juvenile rats by immunofluorescence while monitoring morphological changes in the filiform papillae by laser-scanning microscopy, in transmission mode, of the same sections. No K13 and K14 immunoreactivity was detected on the lingual epithelium of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of a few layers of cuboidal cells. K14 immunoreactivity was first detected on the lingual epithelium of fetuses on E17 and K13 immunoreactivity on E19. The number of layers of cuboidal cells in the lingual epithelium also increased from E17 to E19. K13 and K14 immunoreactivity was distinct at all postnatal stages examined. Although the respective patterns of K13 and K14 immunoreactivity differed as the filiform papillae developed, K13 immunoreactivity was generally evident in the suprabasal cells of the interpapillary cell columns and K14 immunoreactivity was detected in the basal and suprabasal cells of the papillary and interpapillary cell columns. Our newly developed technique for dual localization of antigens should be useful for investigations of very small specimens, such as fetal tissues and organs.