Abstract
The ultrastructural characterization of neuronal compartments in intact tissue labeled with green fluorescent protein (GFP) remains a frequently encountered challenge, despite work establishing photooxidation of GFP in cultured cells. However, most applications require the detection of GFP or GFP fusion proteins expressed in intact tissue. Here, we report that illumination of GFP variants in oxygen-enriched environment reliably generated electron-dense 3,3′-diaminobenzidine (DAB) precipitates in slices from rat brain. The method is applicable to GFP variants tagged to presynaptic proteins as well as to soluble GFP in various brain regions. Serial section scanning electron microscopy was used to examine genetically labeled presynaptic terminals at high resolution and to generate three-dimensional representations of the synapses. Thus, we introduce a generally applicable correlative approach for the identification of presynaptic terminals genetically labeled with green fluorescent proteins in tissue slices and their ultrastructural characterization.
Highlights
Green fluorescent protein (GFP) and its derivatives have been intensely used as genetically encoded reporters for intracellular protein expression or for the examination of protein-protein interactions at the resolution of fluorescence microscopy [1,2]
We show that illumination of presynaptic terminals expressing EGFP or EGFP fusion proteins under carefully controlled conditions generates an amount of reactive oxygen species sufficient to oxidize DAB into electron-dense precipitates, which can be detected in the electron microscope (EM)
The electron-dense material of the DAB precipitate could be readily identified in tissue from the medial nucleus of the trapezoid body (MNTB) allowing us to identify the synaptophysin-2pHluorin overexpressing presynaptic terminals by scanning electron microscopy
Summary
Green fluorescent protein (GFP) and its derivatives have been intensely used as genetically encoded reporters for intracellular protein expression or for the examination of protein-protein interactions at the resolution of fluorescence microscopy [1,2]. One strategy relies on photooxidation of genetically encoded probes resulting in a 3,39diaminobenzidine (DAB) electron-dense product that can be visualized with the EM [8,9,10]. These approaches require high local concentrations of GFP (e.g. Golgi-resident enzyme Nacetylgalactoseaminyltransferase-2 fused to EGFP [8]) and have only been demonstrated in cultured cells. Due to the widely spread use of GFP as a fluorescent protein-tag, GFP-positive cells may need to be analyzed on the ultrastructural level This is challenging because of the limited ability of GFP to produce singlet oxygen [11]. Correlation of fluorescence with electron microscopy often works when looking at the cell soma but may be difficult when small compartments such as presynaptic nerve terminals need to be studied
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