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Methodological approach for the detection of both microdamage and fluorochrome labels in ewe bone and human trabecular bone

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Abstract

The purpose of this study was to adapt various staining methods for the detection of microdamage in human bone, while preserving tetracycline labels. We describe two staining methods using calcein green and xylenol orange, first developed in ewe bone samples and validated in human trabecular bone samples. In ewe bones, we found that calcein green at 0.5 mM concentration diluted in 100% ethanol as well as xylenol orange at 5 mM were the most adequate fluorochromes both to detect microdamage and preserve the double tetracycline labeling. These results were verified in human trabecular bone (iliac crest for the tetracycline label, and vertebral bone for the double labeling). Results obtained in human bone samples were identical to those in ewes, so this combination of fluorochromes is now used in our laboratory.

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Notes

  1. B660-30 JT Baker, Phillipsburg, NJ, USA.

  2. F 6377 Sigma Chemical Co., St Louis, MO, USA.

  3. M1255 Sigma.

  4. A3882 Sigma.

  5. C 0875 Sigma.

  6. 398187 Sigma.

  7. Filter set #20: Excitation BP 546/12, Beam Splitter FT 560, Emission BP 575–640.

  8. Filter set #09: Excitation BP 450–490, Beam Splitter FT 510, Emission LP 515.

  9. Filter set #18: Excitation BP 390–420, Beam Splitter FT 425, Emission LP 450.

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Acknowledgments

The authors wish to acknowledge Dr Anna Bencsik, from histology dept of Anses Lyon laboratory for her assistance in the preparation of the manuscript, and Jean-Paul Roux, from INSERM UMR 1033 for his technical assistance.

Conflict of interest

All the authors state that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. INSERM, UMR1033, 69008, Lyon, France

    Brigitte Burt-Pichat, Hélène Follet, Gwendoline Toulemonde, Monique Arlot, Pierre Delmas & Roland Chapurlat

  2. Université de Lyon, UMR1033, 69008, Lyon, France

    Brigitte Burt-Pichat, Hélène Follet, Gwendoline Toulemonde, Monique Arlot, Pierre Delmas & Roland Chapurlat

  3. INSERM, UMR1033, Faculté de Médecine Lyon Est (domaine Laennec), 7-11 Rue G. Paradin, 69008, Lyon, France

    Hélène Follet

Authors
  1. Brigitte Burt-Pichat
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  2. Hélène Follet
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  3. Gwendoline Toulemonde
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  4. Monique Arlot
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  5. Pierre Delmas
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  6. Roland Chapurlat
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Correspondence to Hélène Follet.

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Burt-Pichat, B., Follet, H., Toulemonde, G. et al. Methodological approach for the detection of both microdamage and fluorochrome labels in ewe bone and human trabecular bone. J Bone Miner Metab 29, 756–764 (2011). https://doi.org/10.1007/s00774-011-0291-7

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  • DOI: https://doi.org/10.1007/s00774-011-0291-7

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