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Improved biocytin labeling and neuronal 3D reconstruction

Nature Protocols volume 7, pages 394–407 (2012)Cite this article

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Abstract

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42–48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

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Figure 1: Flowchart for biocytin labeling.
Figure 2: 'Corkscrew' effect as a result of overly rapid dehydration.
Figure 3: Optical effects in the barrel field of neocortical layer 4 due to different embedding media.
Figure 4: Visibility of cortical lamination in different embedding media.
Figure 5: Resolution of subcellular structures in slices embedded in Moviol and Eukitt.
Figure 6: Shrinkage correction of biocytin-labeled neurons in brain slices.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG; Research Group BaCoFun), the Helmholtz Association and the Helmholtz Alliance for Systems Biology. We thank A. Rollenhagen for help with the EM protocol and T. Abel for critically reading the manuscript.

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Authors and Affiliations

  1. Institute for Neuroscience and Medicine (INM-2), Research Center Jülich, Jülich, Germany

    Manuel Marx, Robert H Günter, Werner Hucko, Gabriele Radnikow & Dirk Feldmeyer

  2. Department of Psychiatry, Psychotherapy and Psychosomatics, RWTH Aachen University, Aachen, Germany

    Dirk Feldmeyer

  3. Jülich Aachen Research Alliance, Translational Brain Medicine (JARA Brain), Aachen, Germany

    Dirk Feldmeyer

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Contributions

M.M. and W.H. performed the experiments; M.M. and R.H.G. analyzed data; M.M., R.H.G., G.R. and D.F. wrote the manuscript; and D.F. and G.R. designed the study.

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Correspondence to Dirk Feldmeyer.

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Marx, M., Günter, R., Hucko, W. et al. Improved biocytin labeling and neuronal 3D reconstruction. Nat Protoc 7, 394–407 (2012). https://doi.org/10.1038/nprot.2011.449

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