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Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes

Nature Protocols volume 8, pages 481–490 (2013)Cite this article

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Abstract

Here we provide a protocol for rapidly labeling different cell types, distinct subcellular compartments and key injury mediators in the spinal cord of living mice. This method is based on the application of synthetic vital dyes to the surgically exposed spinal cord. Suitable vital dyes applied in appropriate concentrations lead to reliable in vivo labeling, which can be combined with genetic tags and in many cases preserved for postfixation analysis. In combination with in vivo imaging, this approach allows the direct observation of central nervous system physiology and pathophysiology at the cellular, subcellular and functional level. Surgical exposure and preparation of the spinal cord can be achieved in less than 1 h, and then dyes need to be applied for 30–60 min before the labeled spinal cord can be imaged for several hours.

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Figure 1: Removal of the meninges.
Figure 2: Concentration dependence of vital dye labeling.
Figure 3: Reliability of relabeling using vital dyes.
Figure 4: Structural dyes.
Figure 5: Functional dyes.
Figure 6: Typical results from the Thy1-YFP-16 mouse.

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Acknowledgements

We thank G. Heitmann for excellent technical assistance and D. Matzek for animal husbandry. Work in M.K.'s laboratory is financed through grants from the European Research Council (ERC Starting Grant), and the Deutsche Forschungsgemeinschaft (DFG; Sonderforschungsbereiche (SFB) 571, SFB 870 and SFB-Tr 128), the German Federal Ministry of Research and Education (Competence Network Multiple Sclerosis) and the 'Verein Therapieforschung für MS-Kranke e.V.' T.M. is supported by the Institute of Advanced Studies (Technische Universität München), the Alexander von Humboldt Foundation, the Center for Integrated Protein Science (Munich), the DFG (SFB 596) and the German Center for Neurodegenerative Disease (DZNE Munich). Work on this project was further supported by the national funding agency ('Bundesministerium für Bildung und Forschung') in the frame of ERA-Net '2-photon imaging' (T.M.) and a grant from the DANA foundation to M.K. and T.M. C.D.S. was supported by the Graduate School of Technische Universität München (TUM-GS).

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Author notes

  1. Elisa Romanelli, Catherine D Sorbara and Ivana Nikić: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Clinical Neuroimmunology, Ludwig-Maximilians Universität München, Munich, Germany

    Elisa Romanelli, Catherine D Sorbara, Ivana Nikić, Athanasios Dagkalis & Martin Kerschensteiner

  2. Biomolecular Sensors, Center for Integrated Protein Sciences (Munich) at the Institute of Neuroscience, Technische Universität München, Munich, Germany

    Catherine D Sorbara & Thomas Misgeld

  3. Institute of Advanced Study, Technische Universität München, Munich, Germany

    Thomas Misgeld

  4. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany

    Thomas Misgeld

  5. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

    Thomas Misgeld & Martin Kerschensteiner

Authors
  1. Elisa Romanelli
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Contributions

M.K., T.M., E.R., C.D.S. and I.N. conceived the experiments. E.R., C.D.S., I.N. and A.D. performed imaging experiments and image analysis. M.K., T.M., E.R., C.D.S. and I.N. wrote the protocol.

Corresponding author

Correspondence to Martin Kerschensteiner.

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Romanelli, E., Sorbara, C., Nikić, I. et al. Cellular, subcellular and functional in vivo labeling of the spinal cord using vital dyes. Nat Protoc 8, 481–490 (2013). https://doi.org/10.1038/nprot.2013.022

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