A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis.
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.
Knott
Graham W
GW
Bio EM Facility, Centre of Interdisciplinary Electron Microscopy, EPFL, Lausanne, Switzerland. Graham.Knott@EPFL.ch
Holtmaat
Anthony
A
Trachtenberg
Joshua T
JT
Svoboda
Karel
K
Welker
Egbert
E
D000818:Animals; D001369:Axons / Q000378:metabolism / Q000648:ultrastructure; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods; D007150:Immunohistochemistry; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008867:Microtomy; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D012984:Software; D013194:Staining and Labeling; D013569:Synapses / Q000648:ultrastructure
D049452:Green Fluorescent Proteins