Abstract
Immuno-electron microscopy (EM) is fundamentally limited by problems related to hindrance of antigenicity in fixed and resin-embedded specimens and destruction of ultrastructure by detergents. Furthermore, immunolabeling is problematic to use with three-dimensional (3D) EM techniques, such as serial block-face scanning EM (SBEM). Here we present an approach for the identification of glutamatergic synaptic vesicles (SVs) in nerve terminals that does not rely on immunolabeling and is compatible with both transmission and 3D EM applications. This technique utilizes heterologous expression of recombinant vesicular glutamate transporter-2 (VGLUT2) fused with mini singlet oxygen generator (miniSOG) and photooxidation of diaminobenzidine (DAB) to label glutamatergic SVs in nerve terminals in both fixed cultured cells and brain tissue.
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References
Shu X et al (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9:e1001041
Steinkellner T et al (2021) Genetic probe for visualizing glutamatergic synapses and vesicles by 3D electron microscopy. ACS Chem Neurosci 12:626–639
Deerinck TJ et al (2018) High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation. J Microsc 270:142–149
Deerinck TJ, Bushong EA, Ellisman MH, Thor A (2022) Preparation of biological tissues for serial block face scanning electron microscopy (SBEM) V.2. protocols.io https://www.protocols.io/view/preparation-of-biological-tissues-for-serial-block-36wgq7je5vk5/v2. https://doi.org/10.17504/protocols.io.36wgq7je5vk5/v2
Hilgenberg LGW, Smith MA (2007) Preparation of dissociated mouse cortical neuron cultures. J Vis Exp 10
Osten P, Cetin A, Komai S, Eliava M, Seeburg PH (2007) Stereotaxic gene delivery in the rodent brain. Nat Protocols 6(1):3166–3173
Los GV et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3:373–382
Martell JD et al (2012) Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy. Nat Biotechnol 30:1143
Masato A et al (2023) DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson’s disease. NPJ Parkinsons Dis 9
Tsang TK et al (2018) High-quality ultrastructural preservation using cryofixation for 3d electron microscopy of genetically labeled tissues. Elife 7
Ganguly A et al (2021) Clathrin packets move in slow axonal transport and deliver functional payloads to synapses. Neuron 109:2884–2901.e7
Acknowledgments
The work presented here was conducted at the National Center for Microscopy and Imaging Research (NCMIR) at UC San Diego directed by Prof. Mark H. Ellisman and is supported by NIH Grants R01GM086197 (D.B.), R01MH120685 (T.S.H and D.B.), R01DA036612 (T.S.H.), 1U24NS120055 (MHE), NSF2014862 (MHE), and R24GM137200 (MHE).
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Flores, A.J. et al. (2024). Photooxidation of Genetically Encoded MiniSOG-Fused VGLUT2 for Identification of Glutamatergic Synapses by Transmission and 3D Electron Microscopy. In: Kukley, M. (eds) New Technologies for Glutamate Interaction. Neuromethods, vol 207. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3742-5_7
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DOI: https://doi.org/10.1007/978-1-0716-3742-5_7
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