Photooxidation-guided ultrastructural identification and analysis of cells in neuronal tissue labeled with green fluorescent protein.

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.