A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis

Abstract

In vivo imaging of green fluorescent protein (GFP)-labeled neurons in the intact brain is being used increasingly to study neuronal plasticity. However, interpreting the observed changes as modifications in neuronal connectivity needs information about synapses. We show here that axons and dendrites of GFP-labeled neurons imaged previously in the live mouse or in slice preparations using 2-photon laser microscopy can be analyzed using light and electron microscopy, allowing morphological reconstruction of the synapses both on the imaged neurons, as well as those in the surrounding neuropil. We describe how, over a 2-day period, the imaged tissue is fixed, sliced and immuno-labeled to localize the neurons of interest. Once embedded in epoxy resin, the entire neuron can then be drawn in three dimensions (3D) for detailed morphological analysis using light microscopy. Specific dendrites and axons can be further serially thin sectioned, imaged in the electron microscope (EM) and then the ultrastructure analyzed on the serial images.

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Figure 1: Finding the location of the imaged cell in the resin embedded sections.
Figure 2: Locating the imaged dendrite in the resin embedded sections.
Figure 3: Trimming and serially sectioning the region of interest.
Figure 4: Electron microscopy and reconstruction of imaged dendrite.

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Acknowledgements

This work was supported by the Swiss National Science Foundation (G.W.K. no. 3100A0-112335 and E.W. no. 310000-108245). The authors would like to thank Stéphanie Rosset for technical help in the development of this protocol.

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G.W.K., J.T.T., K.S. and E.W. conceived the strategy for carrying out the electron microscopy on the imaged neurites. J.T.T., A.H. and K.S. carried out all of the in vivo imaging. K.S. and E.W. provided equipment and reagents. G.W.K. carried out the electron microscopy and wrote the manuscript.

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Correspondence to Graham W Knott.

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Knott, G., Holtmaat, A., Trachtenberg, J. et al. A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat Protoc 4, 1145–1156 (2009). https://doi.org/10.1038/nprot.2009.114

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