Advertisement

Phenotyping Intact Mouse Bones Using Bone CLARITY

  • Jennifer B. Treweek
  • Aidan Beres
  • Nathan Johnson
  • Alon GreenbaumEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2230)

Abstract

The bone is typically studied using traditional histology techniques, that is, serial sectioning and staining. While effective, these techniques are laborious and destructive, as the native 3D environment of the bone is not maintained. Presented here is a bone-clearing methodology, termed Bone CLARITY, which combines published techniques for clearing soft tissues, including delipidation for the removal of light-scattering membranes, hydrogel-embedding for the stabilization of fragile epitopes, heme elution for the reduction of blood-based autofluorescence; as well as specialized steps, including decalcification and progressive refractive index matching, for addressing the unique challenges posed by osseous tissue. This method renders the bone transparent and enables the detailed visualization of an intact tissue specimen at multiple spatial scales.

Key words

Tissue Clearing Bone CLARITY Three-dimensional imaging Light-sheet microscopy Fluorescence microscopy 

References

  1. 1.
    Long F (2012) Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol Cell Biol 13:27–38.  https://doi.org/10.1038/nrm3254CrossRefGoogle Scholar
  2. 2.
    Goltzman D (2002) Discoveries, drugs and skeletal disorders. Nat Rev Drug Discov 1:784–796.  https://doi.org/10.1038/nrd916CrossRefPubMedGoogle Scholar
  3. 3.
    Jacques SL (2013) Optical properties of biological tissues: a review. Phys Med Biol 58:R37–R61.  https://doi.org/10.1088/0031-9155/58/11/R37CrossRefPubMedGoogle Scholar
  4. 4.
    Balani DH, Ono N, Kronenberg HM (2017) Parathyroid hormone regulates fates of murine osteoblast precursors in vivo. J Clin Invest 127:3327–3338.  https://doi.org/10.1172/JCI91699CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Morrison SJ, Scadden DT (2014) The bone marrow niche for haematopoietic stem cells. Nature 505:327–334.  https://doi.org/10.1038/nature12984CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Liu Z, Chen J, Mirando AJ et al (2015) A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis. Sci Signal 8(386):ra71.  https://doi.org/10.1126/scisignal.aaa3792CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pan C, Cai R, Quacquarelli FP et al (2016) Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods 13:859–867.  https://doi.org/10.1038/nmeth.3964CrossRefPubMedGoogle Scholar
  8. 8.
    Chung K, Wallace J, Kim S-Y et al (2013) Structural and molecular interrogation of intact biological systems. Nature 497:332–337.  https://doi.org/10.1038/nature12107CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Yang B, Treweek JB, Kulkarni RP et al (2014) Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158:945–958.  https://doi.org/10.1016/j.cell.2014.07.017CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Treweek JB, Chan KY, Flytzanis NC et al (2015) Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat Protoc 10:1860–1896.  https://doi.org/10.1038/nprot.2015.122CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Renier N, Wu Z, Simon DJ et al (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159:896–910.  https://doi.org/10.1016/j.cell.2014.10.010CrossRefPubMedGoogle Scholar
  12. 12.
    Richardson DS, Lichtman JW (2015) Clarifying tissue clearing. Cell 162:246–257.  https://doi.org/10.1016/j.cell.2015.06.067CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Jing D, Zhang S, Luo W et al (2018) Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res 28:803–818.  https://doi.org/10.1038/s41422-018-0049-zCrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Greenbaum A, Chan KY, Dobreva T et al (2017) Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci Transl Med 9(387):eaah6518.  https://doi.org/10.1126/scitranslmed.aah6518CrossRefPubMedGoogle Scholar
  15. 15.
    Jing D, Yi Y, Luo W et al (2019) Tissue clearing and its application to bone and dental tissues. J Dent Res 98:621–631.  https://doi.org/10.1177/0022034519844510CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Grüneboom A, Hawwari I, Weidner D et al (2019) A network of trans-cortical capillaries as mainstay for blood circulation in long bones. Nat Metab 1:236–250.  https://doi.org/10.1038/s42255-018-0016-5CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Klingberg A, Hasenberg A, Ludwig-Portugall I et al (2017) Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy. J Am Soc Nephrol 28:452–459.  https://doi.org/10.1681/ASN.2016020232CrossRefPubMedGoogle Scholar
  18. 18.
    Tomer R, Ye L, Hsueh B et al (2014) Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9:1682–1697.  https://doi.org/10.1038/nprot.2014.123CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Royer LA, Lemon WC, Chhetri RK et al (2016) Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat Biotechnol 34:1267–1278.  https://doi.org/10.1038/nbt.3708CrossRefPubMedGoogle Scholar
  20. 20.
    Coutu DL, Kokkaliaris KD, Kunz L et al (2017) Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules. Nat Biotechnol 35:1202–1210.  https://doi.org/10.1038/nbt.4006CrossRefPubMedGoogle Scholar
  21. 21.
    Coutu DL, Kokkaliaris KD, Kunz L et al (2018) Multicolor quantitative confocal imaging cytometry. Nat Methods 15:39–46.  https://doi.org/10.1038/nmeth.4503CrossRefPubMedGoogle Scholar
  22. 22.
    Wang Q, Liu K, Yang L et al (2019) BoneClear: whole-tissue immunolabeling of the intact mouse bones for 3D imaging of neural anatomy and pathology. Cell Res 29:870–872.  https://doi.org/10.1038/s41422-019-0217-9CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Jennifer B. Treweek
    • 1
  • Aidan Beres
    • 2
  • Nathan Johnson
    • 2
  • Alon Greenbaum
    • 2
    • 3
    • 4
    Email author
  1. 1.Department of Biomedical EngineeringUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Joint Department of Biomedical EngineeringNorth Carolina State University and University of North Carolina at Chapel HillRaleighUSA
  3. 3.Comparative Medicine InstituteNorth Carolina State UniversityRaleighUSA
  4. 4.Bioinformatics Research CenterNorth Carolina State UniversityRaleighUSA

Personalised recommendations