Iwasaki Shin-Ichi, Aoyagi Hidekazu
Advanced Research Center, School of Life Dentistry at Niigata, The Nippon Dental University, Niigata, Japan.
Methods Mol Biol. 2011;689:229-40. doi: 10.1007/978-1-60761-950-5_14.
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.
我们开发了一种技术，该技术结合了对环氧树脂包埋样品的半超薄切片进行免疫荧光染色以及通过光学显微镜获得的相应微分干涉对比（DIC）图像，可提供有关组织学和细胞结构免疫定位的详细信息。为了证明我们方法的有效性，我们在大鼠舌丝状乳头的形态发生过程中检查了免疫染色的角蛋白13（K13）和III型胶原蛋白（CIII）的免疫荧光以及相应的DIC图像。在出生后第7天和第14天（P7和P14）的幼鼠舌上皮中检测到了对K13和CIII特异的免疫反应性。对K13特异的免疫反应性明显位于乳头间细胞柱的中间层细胞中，而对CIII特异的免疫反应性在舌上皮和舌肌之间的结缔组织纤维中也很明显。DIC图像显示了舌上皮分层鳞状细胞的角化，以及结缔组织下方的肌发生。此外，在舌肌周围的肌内膜和肌束膜中也可识别出对CIII特异的免疫反应性。因此，我们的方法证明了大鼠舌形态发生过程中K13和CIII免疫反应性模式的变化。
