Fluorescence Immunohistochemistry in Combination with Differential Interference Contrast Microscopy

We have developed a technique, using a combination of immunofluorescent staining of semi-ultrathin sections of epoxy resin-embedded samples and the corresponding differential interference contrast (DIC) images obtained by light microscopy that provides detailed information about the immuno-localization of histological and cellular structures. To demonstrate the effectiveness of our method, we examined the immunofluorescence of immuno-stained keratin 13 (K13) and type III collagen (CIII) and the corresponding DIC images during the morphogenesis of filiform papillae on the rat tongue. Immunoreactivity specific for K13 and CIII was detected on the lingual epithelium of juveniles on postnatal days 7 and 14 (P7 and P14). The immunoreactivity specific for K13 was clearly located in the intermediate-layer cells of the interpapillary cell columns, while that specific for CIII was also distinct in the connective-tissue fibers between the lingual epithelium and the lingual muscle. The DIC images revealed the keratinization of the stratified squamous cells of the lingual epithelium and, also, myogenesis beneath the connective tissue. In addition, immunoreactivity specific for CIII was also recognizable in the endomysium and perimysium around the lingual muscle. Thus, our method demonstrated changes in patterns of immunoreactivity of K13 and of CIII during the morphogenesis of the rat tongue.