Demineralized Murine Skeletal Histology

  • Sarah A. Mack
  • Kathleen M. Maltby
  • Matthew J. Hilton
Part of the Methods in Molecular Biology book series (MIMB, volume 1130)


Cartilage and bone are specialized skeletal tissues composed of unique extracellular matrices. Bone, in particular, has a highly calcified or mineralized matrix that makes microtomy and standard histological studies very challenging. Therefore, methods to appropriately fix and decalcify mineralized skeletal tissues have been developed to allow for paraffin processing and standard microtomy. In this chapter, we illustrate methods for tissue grossing, fixation, decalcification, paraffin processing, embedding, sectioning, and routine histological staining of demineralized murine skeletal tissues. We also discuss methods for decalcified frozen sectioning of skeletal tissues with and without the use of a tape-transfer system.


Decalcification Fixation Processing Embedding Sectioning Bone Cartilage 



We would like to thank Ryan Tierney for the initial development of some of these protocols. This work was supported in part by an NIH P30 Core Centers Grant (AR061307) and R01 grants (AR057022 and AR063071) to MJH.


  1. 1.
    Boskey AL (1992) Mineral-matrix interactions in bone and cartilage. Clin Orthop Relat Res 281:244–274Google Scholar
  2. 2.
    Erben RG (1997) Embedding of bone samples in methylmethacrylate: an improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry. J Histochem Cytochem 45(2):307–313CrossRefPubMedGoogle Scholar
  3. 3.
    Merrell GA, Troiano NW, Coady CE, Kacena MA (2005) Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate. Biotech Histochem 80(3–4):139–146CrossRefPubMedGoogle Scholar
  4. 4.
    Belluoccio D, Rowley L, Little CB, Bateman JF (2013) Maintaining mRNA integrity during decalcification of mineralized tissues. PLoS One 8(3):e58154CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Shibata Y, Fujita S, Takahashi H, Yamaguchi A, Koji T (2000) Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues. Histochem Cell Biol 113(3):153–159CrossRefPubMedGoogle Scholar
  6. 6.
    Bourque WT, Gross M, Hall BK (1993) A histological processing technique that preserves the integrity of calcified tissues (bone, enamel), yolky amphibian embryos, and growth factor antigens in skeletal tissue. J Histochem Cytochem 41(9):1429–1434CrossRefPubMedGoogle Scholar
  7. 7.
    Callis GM (2008) “8/Bone.” The theory and practice of histological techniques, 6th edn. Churchill Livingston Elsevier, London, pp 333–348CrossRefGoogle Scholar
  8. 8.
    Callis G, Sterchi D (2002) Animal processing manual. National Society for Histotechnology, Bowie, MDGoogle Scholar
  9. 9.
    Salie R, Li H, Jiang X, Rowe DW, Kalajzic I, Susa M (2008) A rapid, nonradioactive in situ hybridization technique for use on cryosectioned adult mouse bone. Calcif Tissue Int 83(3):212–221CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Sarah A. Mack
    • 1
  • Kathleen M. Maltby
    • 1
  • Matthew J. Hilton
    • 1
  1. 1.University of Rochester Medical CenterRochesterUSA

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