Abstract
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.
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References
Mayor, H. D., Hampton, J. C., Rosario, B. (1961) A simple method for removing the resin from epoxy-embedded tissue. J Cell Biol 9, 909–910.
Haraguchi, C. M., Yokota, S. (2002) Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues. Histochem Cell Biol 117, 81–85.
Litwin, J. A., Yokota, S., Hashimoto, T., Fahimi, H. D. (1984) Light microscopic immunocytochemical demonstration of peroxisomal enzymes in Epon sections. Histochemistry 81, 15–22.
Iwasaki, S., Aoyagi, H., Yoshizawa, H. (2003) Immunohistochemical detection of the expression of keratin 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae. Arch Oral Biol 48, 605–613.
Iwasaki, S., Aoyagi, H., Asami, T. (2006) Expression of keratin 18 in the periderm cells of the lingual epithelium of fetal rats: visualization by fluorescence immunohistochemistry and differential interference contrast microscopy. Odontology 94, 64–68.
Iwasaki, S., Yoshizawa, H., Aoyagi, H. (2006) Immunohistochemical expression of keratin 13 and 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae. Arch Oral Biol 51, 416–426.
Iwasaki, S., Aoyagi, H., Yoshizawa, H. (2007) Immunohistochemical detection of epidermal growth factor and epidermal growth factor receptor in the lingual mucosa of rats during the morphogenesis of filiform papillae. Acta Histochem 109, 37–44.
Iwasaki, S., Aoyagi, H. (2007) Expression of keratin 14 in the basal cells of the lingual epithelium of mice during the morphogenesis of filiform papillae: visualization by fluorescent immunostaining and confocal laser-scanning microscopy in the transmission mode. Odontology 95, 61–65.
Asami, T., Aoyagi, H., Yoshizawa, H., Wanichanon, C., Iwasaki, S. (2008) Immunohistochemical expression of type II collagen in the lingual mucosa of rats during the morphogenesis of the tongue. Arch Oral Biol 53, 622–628.
Aoyagi, H., Asami, T., Yoshizawa, H., Wanichanon, C., Iwasaki, S. (2008) Newly developed technique for dual localization of keratin 13 and 14 by fluorescence immunohistochemistry. Acta Histochem 110, 324–332.
Iwasaki, S., Asami, T., Wanichanon, C., Yoshizawa, H., Aoyagi, H. (2008) Immunohistochemical analysis of type III collagen expression in the lingual mucosa of rats during organogenesis of the tongue. Odontology 96, 12–20.
Shi, S. R., Key, M. E., Karla, K. L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhanced method for immunohistochemical staining based on microwave heating of tissue sections. J Histochem Cytochem 39, 741–748.
Morgan, P. R., Leigh, I. M., Purkis, P. E., Gardner, I. D., Van Muijen, G. N. P., Lane, E. B. (1987) Site variation in keratin expression in human oral epithelia – an immunocytochemical study of individual keratins. Epithelia 1, 31–43.
Barrett, A. W., Selvarajah, S., Franey, S., Wills, K. A., Berkovitz, B. K. B. (1998) Interspecies variations in oral epithelial cytokeratin expression. J Anat 193, 185–193.
Heyden, A., Huitfeldt, H. S., Koppang, H. S., Thrane, P. S., Bryne, M., Brandtzaeg, P. (1992) Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. J Oral Pathol Med 21, 7–11.
Keene, D. R., Sakai, L. Y., Bächinger, H. P., Burgeson, R. E. (1987) Type III collagen can be present on banded collagen fibrils regardless of fibril diameter. J Cell Biol 105, 2393–2402.
Garrone, R., Lethias, C., Guellec, D. (1997) Distibution of minor collagens during skin development. Microsc Res Tech 38, 407–412.
Acknowledgments
The authors thank Professor Sumio Yoshie for his useful advice and they also thank Drs. Hiroyuki Yokosuka and Masahiko Kumakura for their skilled technical assistance. This work was supported by Grants-in-Aid (NDUF-06-04 NDUF-06-21 and NDUF-07-01) from the Nippon Dental University.
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Iwasaki, Si., Aoyagi, H. (2011). Fluorescence Immunohistochemistry in Combination with Differential Interference Contrast Microscopy for Studies of Semi-ultrathin Specimens of Epoxy Resin-Embedded Samples. In: Chiarini-Garcia, H., Melo, R. (eds) Light Microscopy. Methods in Molecular Biology, vol 689. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-950-5_14
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DOI: https://doi.org/10.1007/978-1-60761-950-5_14
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