Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue


High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cells in situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in 11 d.

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Figure 1: Flowchart showing the overview of skeletal tissue processing and imaging protocol.
Figure 2: Skeletal architecture and antigenicity are preserved after tissue processing and immunostaining.
Figure 3: Maintenance of cellular morphology and preservation of antigenicity allows multicolor immunofluorescence.
Figure 4: Bone processing and sectioning described in this protocol allow combinations of multiple fluorescent techniques.
Figure 5: Analysis of genetically expressed fluorescent proteins in skeletal tissue sections.


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We thank M. Schiller for technical assistance and S. Volkery for microscopy. Funding was provided by the Max Planck Society, the University of Münster, the Deutsche Forschungsgemeinschaft (DFG) cluster of excellence 'Cells in Motion', and the European Research Council (AdG 339409 AngioBone).

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A.P.K., S.K.R. and R.H.A. designed experiments, contributed to the development of methodology and wrote the paper. A.P.K. and S.K.R. directed A.S. and carried out the experiments.

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Correspondence to Anjali P Kusumbe or Saravana K Ramasamy or Ralf H Adams.

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The authors declare no competing financial interests.

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Kusumbe, A., Ramasamy, S., Starsichova, A. et al. Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue. Nat Protoc 10, 1904–1914 (2015).

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