ARTICLE |
Correspondence to: Antonio Nanci, Dépt. de Stomatologie, Faculté de Médecine Dentaire, Université de Montréal, CP 6138, Succ. Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail: nancia@ere.umontreal.ca
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Summary |
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Vimentin, an intermediate filament component, has been identified in many mesenchymal cells by a variety of LM and EM immunolabeling techniques. In our study, several tissue-processing conditions and monoclonal and polyclonal antibodies against vimentin were screened for immunostaining of rat incisor odontoblasts. Using postembedding colloidal gold immunocytochemistry, we were unable to detect any convincing vimentin antigenicity in these cells, but one of the monoclonal antibodies (V9-S) unexpectedly resulted in intense labeling over intra- and extracellular compartments that normally are strongly immunoreactive with anti-amelogenin antibodies. Blocking experiments showed that V9-S binding was competed by anti-amelogenin antibody. Immunoblots indicated that enamel proteins reacted with this anti-vimentin antibody after fixation with glutaraldehyde. These data suggest that the observed immunoreaction is directed against an epitope apparently created by crosslinking of enamel proteins during fixation. Although the labeling cannot be considered specific, it is nevertheless selective because it is very precisely localized over compartments containing enamel proteins and shows no binding to other calcified dental tissues, including dentin and bone. The V9-S antibody can therefore be used as a reliable probe to identify the presence and distribution of amelogenins in fixed tissues. (J Histochem Cytochem 47:12371245, 1999)
Key Words: vimentin, immunocytochemistry, immunoblotting, amelogenesis, enamel proteins, incisor, rat
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Introduction |
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Tooth development is mediated by reciprocal epithelialmesenchymal interactions between neural crest-derived ectomesenchymal cells and the oral epithelium (reviewed in
The initial objective of this study was to characterize the frequency with which odontoblast processes invaginate into differentiating ameloblasts. Because these small cytoplasmic extensions are difficult to identify and distinguish morphologically in cross-sections, we originally planned to use vimentin antibodies to "tag" and make them more visible. This intermediate filament protein is typically associated with mesenchymal cells (
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Materials and Methods |
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Tissue Preparation for Immunocytochemistry
Male Wistar rats (Charles Rivers Canada; St-Constant, Quebec, Canada) weighing approximately 100 g were anesthetized with chloral hydrate (Sigma Chemical; St Louis, MO) and perfused through the ascending aorta with lactated Ringer's solution (Abbott; Montreal, Quebec, Canada) for 30 sec and then with either 4% paraformaldehyde containing 0.1% glutaraldehyde or 1% or 2.5% glutaraldehyde for 20 min. All fixatives were buffered with 0.08 M sodium cacodylate containing 0.05% CaCl2 at pH 7.3. After perfusion, the hemimandibles were removed and immersed in their respective fixative overnight at 4C. Some hemimandibles were decalcified in 4.13% disodium EDTA at 4C (
Thin sections of the tissues were cut with a diamond knife on a Reichert Ultracut E ultramicrotome, mounted on 200-mesh nickel grids having a carbon-coated Formvar film, and processed for postembedding colloidal gold immunolabeling (reviewed in
Tissue Processing for Embedding
Epoxy Resin.
Some incisor samples fixed in 1% or 2.5% glutaraldehyde and postfixed in reduced osmium were dehydrated in a graded series of acetone (30100%). They were then infiltrated for 812 hr with a 1:1 and 2:1 mixture of Epon substitute resin (Meca Laboratories; Montreal, QC, Canada) and pure acetone, followed by pure resin for 5 hr under vacuum. Tissues were embedded in a fresh mixture of pure resin and were polymerized for 2 days at 60C.
LR White. Other tooth samples fixed in 1% glutaraldehyde and postfixed in reduced osmium were dehydrated in graded concentrations of ethanol (30100%) and infiltrated with a 1:1 mixture of LR White resin (hard grade; Bio-Rad Laboratories, WatfordHertfordshire, UK) and absolute ethanol for 1 hr, followed by three 1-hr changes in pure LR White resin, all at room temperature (RT). The tissues were then embedded in pure LR White in gelatin capsules and the resin was polymerized at 48C for 2 days.
Lowicryl K4M. Nonosmicated tissue segments from animals fixed in paraformaldehyde/glutaraldehyde mixture were processed for embedding in a cryosubstitution apparatus (CS auto; ReichertJung, Wien, Austria). They were dehydrated in 30% methanol at 0C, in 50% methanol at -20C, and in 75% methanol at -35C for 30 min each, and in 90% methanol for 60 min at -35C. The tissues were then infiltrated at -35C for periods of 60 min with 1:1 and 2:1 mixtures of Lowicryl K4M (Chemische Werke Lowi; Waldkraiburg, Germany) and 90% methanol, followed by pure Lowicryl K4M overnight. They were then embedded in pure resin in plastic molds and polymerized for 2 days at -35C using the UV illuminator arrangement of the CS auto apparatus.
Immunocytochemical Procedures
Antibody Incubation.
All immunocytochemical procedures were performed at RT. Grid-mounted sections of osmicated tissues embedded in Epon or LR White were pretreated with saturated sodium metaperiodate for 1 hr (
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For controls, grid-mounted sections were incubated with secondary antibody only (see Table 1) followed by protein Agold, or with protein Agold alone.
Evaluation of the Specificity of the Anti-vimentin Immunolabeling by Competitive Binding
LectinGold Cytochemistry.
Tissue sections of Lowicryl-embedded samples were floated on a drop of wheat germ agglutinin (WGA) at a concentration of 25 µg/ml for 1 hr, rinsed with PBS, and incubated for 30 min with ovomucoidgold (8-nm) prepared as described in
Immunolabeling with Antibody to Amelogenin. Sections of nonosmicated tissues were first incubated with AMEL antibody (see Table 1) for 1 hr, rinsed with PBS, and floated on PBS1% ovalbumin (5 min), followed by V9-S antibody (diluted 1:10) for 1 hr. To exclude the possibility that steric hindrance of the AMEL IgG might affect the labeling pattern of V9-S antibody, nonosmicated sections were floated on WGA (25 µg/ml) for 1 hr before incubation with clone V9-S (dilution 1:10) for 1 hr. All sections were then rinsed and incubated with protein Agold as described above.
Immunoblotting.
Purified vimentin (Sigma), human fibroblast lysate, and proteins extracted from freeze-dried secretory stage enamel samples (
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Results |
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Four antibodies against vimentin were tested on plastic sections of rat incisors for their ability to immunoreact with odontoblasts. None showed any significant immunolabeling in these cells. However, some of these antibodies were found to localize over intra- and extracellular compartments of ameloblasts previously shown to contain amelogenins (
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Antibody V9-BM gave almost no immunolabeling over ameloblasts, regardless of resin type. The density of gold particles over enamel was also highly variable in relation to embedding media and fixatives used (Table 2). Antibody V-poly gave only weak immunolabeling over the enamel matrix, whereas antibody V-3B4 showed no immunoreactivity over intra- and extracellular amelogenin-containing compartments (Table 2).
Sections incubated with AMEL showed labeling comparable to that of V9-S (Figure 6A and Figure 6B). Competitive incubations with AMEL followed by V9-S resulted in significant abolition of the labeling normally obtained with V9-S (Figure 6C). Sections of Lowicryl K4M-embedded samples incubated with WGA followed by ovomucoidgold likewise showed many gold particles over secretory granules and enamel (Figure 7A). Lectin binding sites were conspicuously concentrated at enamel growth sites (Figure 7A). However, incubation of sections with WGA before labeling with V9-S did not affect binding of the antibody to the tissue sections, as did AMEL (compare Figure 6C and Figure 7B).
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Unfixed secretory stage enamel proteins incubated with V9-S were unreactive. In constrast, purified vimentin and proteins extracted from cultured human fibroblasts showed strong immunostaining (Figure 8). However, when the enamel proteins were fixed with 1% glutaraldehyde before staining with the antibody, immunoreactive bands below 31 kD were revealed. The AMEL antibody did not significantly stain vimentin or any proteins in extracts from cultured human fibroblasts in fixed blots (Figure 8). Faintly stained bands were observed near 60 kD in blots of unfixed vimentin and enamel extracts but not in fibroblast extracts (Figure 8).
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Discussion |
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Vimentin has been detected in various mesenchymal cells types of the tooth organ, including odontoblasts (
A variety of monoclonal and polyclonal antibodies against enamel proteins have been used over the years to elucidate the process of amelogenesis (reviewed in
There are two major families of enamel proteins, amelogenins and nonamelogenins (reviewed in
Monoclonal antibodies are directed against narrow determinants consisting of a few amino acids that can recur on several proteins and peptides. Therefore, it is not surprising that some monoclonals yield labelings on totally unrelated proteins from different tissues. In this regard, a monoclonal antibody against a monocytemacrophage protein has similarly been found to bind to enamel proteins of less than 30 kD (
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Acknowledgments |
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Supported by grants from Calcinfonden, the Danish Dental Association (KJ), and the Medical Research Council of Canada (AN,CES).
We wish to thank Dr Wei-Yu Chen for preparing the Western blots used in Figure 8. We are also grateful to Merete Andersen and Micheline Fortin for technical assistance and to Sylvia Zalzal for preparing the protein Agold complexes used for labeling.
Received for publication April 9, 1999; accepted May 18, 1999.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bendayan M (1995a) Possibilities of false immunocytochemical results generated by the use of monoclonal antibodies: the example of the anti-proinsulin antibody. J Histochem Cytochem 43:881-886
Bendayan M (1995b) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1-163[Medline]
Bendayan M, Zollinger M (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein Agold technique. J Histochem Cytochem 31:101-109[Abstract]
Bradbeer JN, Virdi AS, Serre CM, Beresford JN, Delmas PD, Reeve J, Triffitt JT (1994) A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: implications for the identification of cells of the osteoblastic lineage in vitro. J Bone Miner Res 9:1221-1228[Medline]
Byers MR, Sugaya A (1995) Odontoblast processes in dentin revealed by fluorescent Di-I. J Histochem Cytochem 43:159-168
Chen W-Y, Nanci A, Smith CE (1995) Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins. Calcif Tissue Int 57:145-151[Medline]
Davidson RM (1994) Neural form of voltage-dependent sodium current in human cultured dental pulp cells. Arch Oral Biol 39:613-620[Medline]
Fausser JL, Lesot H, Zidan G, Ruch J-V (1990) Characterization of a mouse monoclonal antibody to vimentin by indirect immunofluorescence microscopy and immunoblotting. J Biol Buccale 18:29-33[Medline]
Frank RM, Nalbandian J (1989) Development of dentine and pulp. In Oksche A, Vollrath L, eds. Handbook of Microscopic Anatomy. Vol V/6. Teeth. Berlin, Springer-Verlag, 73-171
Frens G (1973) Controlled nucleation for the regulation of particle size in monodispersed gold suspensions. Nature Phys Sci 241:20-22
Inoue T, Chen SH, Usuda J, Morohushi Y, Shimono M (1992) Osteogenic activity of cells from dental pulp, periodontal ligament, bone marrow and muscle in vitro: an ultrastructural study and alkaline-phosphatase activity. Bull Tokyo Dent Coll 33:7-12[Medline]
Kallenbach E (1971) Electron microscopy of the differentiating rat incisor ameloblast. J Ultrastruct Res 35:508-531[Medline]
Kallenbach E (1976) Fine structure of differentiating ameloblasts in the kitten. Am J Anat 145:283-318[Medline]
Kallenbach E, Piesco NP (1978) The changing morphology of the epithelium-mesenchyme interface in the differentiation zone of growing teeth of selected vertebrates and its relationship to possible mechanisms of differentiation. J Biol Buccale 6:229-240[Medline]
Kameda Y (1996a) Ultrastructural immunogold localization of vimentin and S-100 protein in guinea pig pars tuberalis. J Histochem Cytochem 44:511-518
Kameda Y (1996b) Immunoelectron microscopic localization of vimentin in sustentacular cells of the carotid body and the adrenal medulla of guinea pigs. J Histochem Cytochem 44:1439-1449[Abstract]
Katchburian E, Burgess AMC (1977) Fine structure of contacts between ameloblasts and odontoblasts in the rat tooth germ. Arch Oral Biol 22:551-553[Medline]
Lazarides E (1982) Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem 51:219-250[Medline]
Lesot H, Meyer JM, Ruch J-V, Weber K, Osborn M (1982) Immunofluorescent localization of vimentin, prekeratin and actin during odontoblast and ameloblast differentiation. Differentiation 21:133-137[Medline]
Lester KS (1970) On the nature of "fibrils" and tubules in developing enamel of the oppossum, Didelphis marsupialis. J Ultrastruct Res 30:64-77[Medline]
Lombardi T, Samson J, Bernard JP, Di Felice R, Fiore-Donno G (1992a) Comparative immunohistochemical analysis between jaw myxoma and mesenchymal cells of tooth germ. Pathol Res Pract 188:141-144[Medline]
Lombardi T, Samson J, Mühlhauser J, Fiore-Donno G, Maggiano N, Castellucci M (1992b) Expression of intermediate filaments and actins in human dental pulp and embryonic dental papilla. Anat Rec 234:587-592[Medline]
Lyn P, FioreDonno G, Lombardi T (1991) The connective tissue cells of human dental pulp: an histologic and immunohistochemical study. Bull Group Int Res Sci Stomatol Odontol 34:133-137
Meyer JM, Fabre M, Staubli A, Ruch JV (1977) Relations cellulaires au cours de l'odontogenèse. J Biol Buccale 5:107-119[Medline]
Nakamura M, Kindaichi K, Kagayama M (1991) Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte-macrophage protein recognized by mouse monoclonal antibody MOMA-2. Arch Oral Biol 36:619-622[Medline]
Nanci A, Ahluwalia JP, Zalzal S, Smith CE (1989) Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor. J Histochem Cytochem 37:1619-1633[Abstract]
Nanci A, Slavkin HC, Smith CE (1987) Application of high-resolution immunocytochemistry to the study of the secretory, resorptive, and degradative functions of ameloblasts. Adv Dent Res 1:148-161[Medline]
Nanci A, Smith CE (1992) Development and calcification of enamel. In Bonucci E, ed. Calcification in Biological Systems. Boca Raton, FL, CRC Press, 313-343
Nanci A, Zalzal S, Lavoie P, Kunikata M, Chen W-Y, Krebsbach PH, Yamada Y, Hammarström L, Simmer JP, Fincham AG, Snead ML, Smith CE (1998) Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors. J Histochem Cytochem 46:911-934
Neiss WF (1984) Electron staining of the cell surface coat by osmium-low ferrocyanide. Histochemistry 80:231-242[Medline]
Papagerakis P, Peuchmaur M, Hotton D, Ferkdadji L, Delmas P, Sasaki S, Tagaki T, Berdal A (1999) Aberrant gene expression in epithelial cells of mixed odontogenic tumors. J Dent Res 78:20-30[Abstract]
Robinson C, Kirkham J, Brookes SJ, Bonass WA, Shore RC (1995) The chemistry of enamel development. Int J Dev Biol 39:145-152[Medline]
Ruch JV, Lesot H, BegueKirn C (1995) Odontoblast differentiation. Int J Dev Biol 39:51-68[Medline]
Ruch J-V, Lesot H, KarcherDjuricic V, Meyer JM, Mark M (1983) Epithelial-mesenchymal interactions in tooth germs: mechanisms of differentiation. J Biol Buccale 11:173-193[Medline]
Sasaki T (1990) Cell biology of tooth enamel formation. In Myers HM, ed. Monographs in Oral Science. Basel, Karger, 1-204
Seux D, Couble ML, Hartmann DJ, Gauthier JP, Magloire H (1991) Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium hydroxide-containing cement. Arch Oral Biol 36:117-128[Medline]
Shapiro F, Cahill C, Malatantis G, Nayak RC (1995) Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique. Anat Rec 241:39-48[Medline]
Sigal MJ, Aubin JE, Ten Cate AR (1985) An immunocytochemical study of the human odontoblast process using antibodies against tubulin, actin, and vimentin. J Dent Res 64:1348-1355[Abstract]
Silva DG, Kailis DG (1972) Ultrastructural studies on the cervical loop and the development of the amelo-dentinal junction in the cat. Arch Oral Biol 17:279-289[Medline]
Sisca RF, Provenza DV (1972) Initial dentin formation in human deciduous teeth. An electron microscope study. Calcif Tissue Res 9:1-16[Medline]
Slavkin HC (1990) Molecular determinants of tooth development: a review. Crit Rev Oral Biol Med 1:1-16[Medline]
Slavkin HC, Bringas PJ (1976) Epithelial-mesenchymal interactions during odontogenesis. IV. Morphological evidence for direct heterotypic cell-cell contacts. Dev Biol 50:428-442[Medline]
Smith CE, Pompura JR, Borenstein S, Fazel A, Nanci A (1989) Degradation and loss of matrix proteins from developing enamel. Anat Rec 224:292-316[Medline]
Smith CE, Nanci A (1996) The protein dynamics of amelogenesis. Anat Rec 245:219-234[Medline]
Steinert PM, Roop DR (1988) Molecular and cellular biology of intermediate filaments. Annu Rev Biochem 57:593-625[Medline]
Thesleff I, Vaahtokari A, Vainio S, Jowett A (1996) Molecular mechanisms of cell and tissue interactions during early tooth development. Anat Rec 245:151-161[Medline]
Van de Klundert FAJM, Raats JMH, Bloemendal H (1993) Intermediate filaments: regulation of gene expression and assembly. Eur J Biochem 214:351-366[Medline]
Warshawsky H, Moore G (1967) A technique for the fixation and decalcification of rat incisors for electron microscopy. J Histochem Cytochem 15:542-549[Medline]
Webb PP, Moxham BJ, Ralphs JR, Benjamin M (1995) Cytoskeleton of the mesenchymal cells of the rat dental papilla and dental pulp. Connect Tissue Res 32:71-76[Medline]
Wise GE, Lin F, Fan W (1992) Culture and characterization of dental follicle cells from rat molars. Cell Tissue Res 267:483-492[Medline]