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Title

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

Authors
Knott, Graham W.
Trachtenberg, Joshua T.
Svoboda, Karel
Welker, Egbert
Published in Nature Protocols. 2009, vol. 4, no. 8, p. 1145-1156
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.
Keywords AnimalsAxons/metabolism/ultrastructureDendrites/metabolism/ultrastructureGreen Fluorescent Proteins/analysis/genetics/metabolismImaging, Three-Dimensional/methodsImmunohistochemistryMiceMicroscopy, ConfocalMicroscopy, Electron, TransmissionMicrotomyNeurons/cytology/metabolism/ultrastructureSoftwareStaining and LabelingSynapses/ultrastructure
Identifiers
PMID: 19617886
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Research group Les circuits neuronaux dans le néocortex des mammifères (855)
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KNOTT, Graham W. et al. A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. In: Nature Protocols, 2009, vol. 4, n° 8, p. 1145-1156. https://archive-ouverte.unige.ch/unige:5399

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Deposited on : 2010-03-11

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