Staining and embedding the whole mouse brain for electron microscopy

Abstract

The development of methods for imaging large contiguous volumes with the electron microscope could allow the complete mapping of a whole mouse brain at the single-axon level. We developed a method based on prolonged immersion that enables staining and embedding of the entire mouse brain with uniform myelin staining and a moderate preservation of the tissue's ultrastructure. We tested the ability to follow myelinated axons using serial block-face electron microscopy.

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Figure 1: Whole mouse brain stained with wbPATCO and embedded with Quetol.
Figure 2: Traceability analysis of eight regions of interest (ROIs) in the mouse brain.
Figure 3: Analysis of axon morphological diversity and tracing error rate.

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Acknowledgements

We thank I. Sonntag and M. Helmstaedter for help with traceability analysis, S. Hillmer and U. Mersdorf for help with transmission electron microscopy, K. Briggman, S.K. Mikula and A. Scherbarth for help with staining procedures, B. Titze for help with conductive coating, J. Tritthardt for developing electronic circuits and M. Mueller for help with scanning electron microscopy–related software. We also thank the following student tracers: M. Diemer, C. Domnick, J. Hanne, P. Hofmann, A. Ivanova, H. Jakobi, A. Klein, J. Löffler, J. Nassel, J. Trendel and P. Weber. This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG).

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Authors

Contributions

S.M. and W.D. designed the study and devised the analysis; S.M. carried out the experiments; J.B. and W.D. devised the aberration correction algorithm; S.M. analyzed the data; and S.M. and W.D. wrote the paper.

Corresponding authors

Correspondence to Shawn Mikula or Winfried Denk.

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Competing interests

W.D. receives license income for 3View serial block-face electron microscopy technology from Gatan.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1–3 and Supplementary Protocol (PDF 663 kb)

Fly-through of a subregion from the internal capsule region of interest.

Voxel size is 40 nm isotropic (MOV 24047 kb)

Rotation animation of axon tracings from the ventroposterolateral nucleus of the dorsal thalamus.

Nodes of Ranvier are indicated by small gray spheres (MOV 17598 kb)

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Mikula, S., Binding, J. & Denk, W. Staining and embedding the whole mouse brain for electron microscopy. Nat Methods 9, 1198–1201 (2012). https://doi.org/10.1038/nmeth.2213

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