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.

Ultrastructural and Functional Properties of a Giant Synapse Driving the Piriform Cortex to Mediodorsal Thalamus Projection

“…Via a juxtaposed double-barrel stimulation-electrode we evoked presynaptic transmitter release in a repetitive stimulation paradigm from identified PIR-MD giant synapses. Furthermore, we used the photooxidation method (Horstmann et al, 2013 ) to identify and reconstruct electron microscopic 3D models of PIR synapses. According to the classification criteria proposed by Sherman and Guillery ( 2006 ), a proportion of PIR-MD synapses fulfills the requirements in all measured parameters to be classified as “class I” synapses.…”

Directed evolution of APEX2 for electron microscopy and proximity labeling

“…Consequently, APEX2 could be used for EM studies and proteomic tagging experiments not previously possible with APEX. As an EM reporter, APEX2 is easier to use than other tags (miniSOG, 20 ReAsH, 21 fluorescent proteins 22 , 23 ) and can provide staining across large fields of view without special equipment, because contrast generation does not require light. The directness of the approach makes it an attractive alternative to indirect assays such as subcellular fractionation followed by protease accessibility tests or western blotting to examine the sub-organellar localization and membrane topology 4 of important proteins.…”

Current Status of Automatic Serial Sections for 3D Electron Microscopy

“…The group developed a new APEX tag ("engineered APX") with the target protein gene and 3,3'-diaminobenzidine (DAB) oxidation to make it visible by EM (Martell et al, 2012). At the Heidelberg Institute, the 3D structure of specific presynaptic terminals was reconstructed by labeling the neurons via the fusion of high-density DAB with GFP-tagging; the results were published in PLOS One (Horstmann et al, 2013). Researchers at the Harvard University used viral vectors to utilize electrondense peroxidases for staining specific neurons.…”