Skip to main content

Advertisement

SpringerLink
Log in

Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following or bypassing fixation in 4% paraformaldehyde, these sections were decalcified in 5% EDTA for 0-5 min. Before decalcification, marked radiopacity of bone matrix was observed in contact microradiography (CMR) images, and electron probe microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In fixed and unfixed sections without decalcification, immunolocalization of bone matrix proteins were almost restricted to osteoid. After 1 min of decalcification, reduced radiopacity was apparent in the CMR images, and less phosphorus and calcium was observed by EPMA, which completely disappeared by 5 min decalcification. After 3-5 min of decalcification, unfixed sections showed that these proteins were immunolocalized in bone matrix, but were not detectable in osteoid. However, fixed sections demonstrated that these were found in both bone matrix and osteoid. The present findings suggest that bone matrix proteins are embedded in calcified matrix which is separated from the aqueous environment and that they hardly move, probably due to firm bonding with each other. In contrast, matrix proteins in osteoid are subject to loss after decalcification because they may be bound to scattered apatite crystals, not to each other.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Arafat HA, Wein AJ, Chacko S (2002) Osteopontin gene expression and immunolocalization in the rabbit urinary tract. J Urol 167:746–752

    Article  CAS  PubMed  Google Scholar 

  • Baker JR, Hew H, Fishman WH (1958) The use of a chloral hydrate formaldehyde fixative solution in enzyme histochemistry. J Histochem Cytochem 6:244–250

    CAS  PubMed  Google Scholar 

  • Begue-Kirn C, Smith AJ, Loriot M, Kupferle C, Ruch JV, Lesot H (1994) Comparative analysis of TGF beta s, BMPs, IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during normal and in vitro-induced odontoblast differentiation. Int J Dev Biol 38:405–420

    CAS  PubMed  Google Scholar 

  • Beninati S, Senger DR, Cordella-Miele E, Mukherjee AB, Chackalaparampil I, Shanmugam V, Singh K, Mukherjee BB (1994) Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin. J Biochem 115:675–682

    CAS  PubMed  Google Scholar 

  • Bosshardt DD, Nanci A (1998) Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells. J Histochem Cytochem 46:135–142

    CAS  PubMed  Google Scholar 

  • Bronckers AL, Farach-Carson MC, Van Waveren E, Butler WT (1994) Immunolocalization of osteopontin, osteocalcin, and dentin sialoprotein during dental root formation and early cementogenesis in the rat. J Bone Miner Res 9:833–841

    CAS  PubMed  Google Scholar 

  • Fink S (1992) A solvent-free coating-procedure for the improved preparation of cryostat section in light microscope histochemistry. Histochemistry 97:243–246

    Article  CAS  PubMed  Google Scholar 

  • Fukase Y (1997) Immunohistochemical staining of human teeth, and production of monoclonal antibodies against cementum. J Osaka Dent Univ 31:1–9

    CAS  PubMed  Google Scholar 

  • Hammarstrom L (1986) Autoradiography and histochemistry of mineralized tissues by means of the Ullberg freeze-sectioning technique. Upsala J Med Sci 91:239–243

    CAS  PubMed  Google Scholar 

  • Helder MN, Bronckers AL, Woltgens JH (1993) Dissimilar expression patterns for the extracellular matrix proteins osteopontin (OPN) and collagen type I in dental tissues and alveolar bone of the neonatal rat. Matrix 13:415–425

    CAS  PubMed  Google Scholar 

  • Hill EL, Elde R (1990) An improved method for preparing cryostat sections of non-decalcified bone for multiple use. J Histocem Cytochem 38:443–448

    CAS  Google Scholar 

  • Hoshi K, Ejiri S, Ozawa H (2001) Organic components of crystal sheaths in bones. J Electron Microsc 50:33–40

    Article  CAS  Google Scholar 

  • Hunter GK, Hauschka PV, Poole AR, Rosenberg LC, Goldberg HA (1996) Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 317:59–64

    CAS  PubMed  Google Scholar 

  • Kaartinen MT, EL-Maadawy S, Rasanen NH, Mckee MD (2002) Tissue transglutaminase and its substrates in bone. J Bone Miner Res 17:2161–2173

    CAS  PubMed  Google Scholar 

  • Kawamoto T (2003) Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol 66:123–143

    Article  PubMed  Google Scholar 

  • Kawamoto T, Shimizu M (2000) A method for preparing 2- to 50-μm-thick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol 113:331–339

    CAS  PubMed  Google Scholar 

  • Macneil RL, Sheng N, Strayhorn C, Fisher LW, Somerman MJ (1994) Bone sialoprotein is localized to the root surface during cementogenesis. J Bone Miner Res 9:1597–1606

    CAS  PubMed  Google Scholar 

  • Mark MP, Butler WT, Prince CW, Finkelman RD, Ruch JV (1988) Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. Differentiation 37:123–136

    CAS  PubMed  Google Scholar 

  • McElroy HH, Shin M-S, Parfitt AM (1993) Producing frozen sections of calcified bone. Biotech Histochem 68:50–55

    CAS  PubMed  Google Scholar 

  • McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A (1993) Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and alpha 2HS-glycoprotein) proteins in rat bone. J Bone Miner Res 8:485–496

    CAS  PubMed  Google Scholar 

  • Miyazaki Y, Setoguchi M, Yoshida S, Higuchi Y, Akizuki S (1990) The mouse osteopontin gene. Expression in monocytic lineages and complete nucleotide sequence. J Biol Chem 265:14432–14438

    Google Scholar 

  • Mukai K, Yoshimura S, Anzai M (1986) Effects of decalcification on immunoperoxidase staining. Am J Surg Pathol 10:413–719

    CAS  PubMed  Google Scholar 

  • Mullink H, Henzen-Logmans SC, Tadama TM, Mol JJ, Meijer CJ (1985) Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies. J Histochem Cytochem 33:1103–1109

    CAS  PubMed  Google Scholar 

  • Nanci A (2003) Ten Cate’s oral histology: development, structure, and function, 6th edn. Mosby, St Louis, pp 61–71

    Google Scholar 

  • Patarca R, Freeman GJ, Sing RP, Wei FY, Durfee T, Blottner F, Regnier DC, Kozak CA, Mock BA, Morse HC (1986) Structual and functional studies of the early T-lymphocyte activation 1 (Eta-1) gene. Definition of a novel T-cell dependent response associated with genetic resistance to bacterial injection. J Exp Med 170:145–161

    Article  Google Scholar 

  • Shimada M, Watanabe M (1995) Recent progress in whole-body radiography. Cell Mol Biol 41:39–48

    CAS  PubMed  Google Scholar 

  • Takeshita N, Kuwahara S, Shirasuga H, Akiba M, Nagai N (1983) The influence of decalcifying solution on immunoperoxidase staining (PAP) in paraffin section. Jpn J Oral Biol 25:1134–1135

    CAS  Google Scholar 

  • Tripp EJ, Mackay EH (1972) Silver staining of bone prior to decalcification for quantitative determination of osteoid in sections. Stain Technol 47:129–136

    CAS  PubMed  Google Scholar 

  • Van Noorden CJ, Vogels IM (1986) Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions. Histochemistry 86:127–133

    Article  CAS  PubMed  Google Scholar 

  • Van Noorden CJ, Vogels IM, Van Wering ER (1989) Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia. Histochemistry 92:313–318

    Article  CAS  PubMed  Google Scholar 

  • Van Den Munckhof RJM, Vreeling-Sinderarova H, Schellens JPM, Frederiks WM (1994) Localization of uric acid oxidase activity in core and matrix of peroxisomes as detected in unfixed cryostat sections of rat liver unfixed. J Histochem Cytochem 42:177–183

    CAS  PubMed  Google Scholar 

  • Villanueva AR, Mehr LA (1977) Modifications of the Goldner and Gomori one-step Trichrome stains for plastic-embedded thin sections of bone. Am J Med Technol 43:536–538

    CAS  PubMed  Google Scholar 

  • Wakisaka S (1986) Immunohistochemical study on substance P in rat molar pulp and periodontal tissues. I. Effect of decalcifying agents on substance P-like immunoreactivity. J Osaka Univ Dental Soc 31:203–208

    CAS  Google Scholar 

  • Yoshiki S (1973) A simple histological method for identification of osteoid matrix in decalcified bone. Stain Technol 48:233–238

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Dr. L.W. Fisher (National Institute of Dental Research, National Institute of Health) and Dr. M. Fukae (Department of Biochemistry, Tsurumi University School of Dental Medicine) for kindly providing the antibodies against BSP and OPN. This work was supported, in part, by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author information

Authors and Affiliations

  1. Department of Oral Histology, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano, 399-0781, Japan

    Akihiro Hosoya, Noriyuki Sahara & Hidehiro Ozawa

  2. Department of Menicon Cartilage and Bone Regeneration, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Kazuto Hoshi

  3. Institute for Dental Science, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano, 399-0781, Japan

    Noriyuki Sahara, Tadashi Ninomiya, Syoji Akahane & Hidehiro Ozawa

  4. RI Research Institute, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa, 230-8501, Japan

    Tadafumi Kawamoto

Authors
  1. Akihiro Hosoya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kazuto Hoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Noriyuki Sahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tadashi Ninomiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Syoji Akahane
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tadafumi Kawamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hidehiro Ozawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Akihiro Hosoya.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hosoya, A., Hoshi, K., Sahara, N. et al. Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections. Histochem Cell Biol 123, 639–646 (2005). https://doi.org/10.1007/s00418-005-0791-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-005-0791-4

Keywords

Search

Navigation