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Practical Guidelines for Two-Color SMLM of Synaptic Proteins in Cultured Neurons

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Single Molecule Microscopy in Neurobiology

Part of the book series: Neuromethods ((NM,volume 154))

Abstract

The application of single-molecule localization microscopy (SMLM) to the study of synaptic proteins has shown that the postsynaptic density (PSD) is organized heterogeneously in subsynaptic domains (SSDs) that are thought to play important roles in neurotransmission and synaptic plasticity. However, the dense packing of neurotransmitter receptors and scaffold proteins at synapses, together with the small total number of target molecules, makes SMLM of synaptic components particularly challenging. Here, we discuss the technical difficulties of SMLM imaging that are specific to synapses. We present a method for dual-color direct stochastic optical reconstruction microscopy (dSTORM) of two inhibitory synaptic proteins, the glycine receptor (GlyR) and the scaffold protein gephyrin (GPHN), highlighting strategic choices and practical solutions for imaging quality control. Our aim is to provide biologists with guidelines for the implementation of two-color dSTORM imaging of synaptic proteins from sample preparation to data analysis.

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Acknowledgment

We thank Manuel Maidorn and Felipe Opazo for the illustrations in Fig. 2 [55], and Ignacio Izeddin for Fig. 4. Our research is funded by grants (to Antoine Triller, IBENS, Paris) from the Agence Nationale de la Recherche (ANR-12-BSV4-0019-01, ANR-11-IDEX-0001-02, ANR-10-LABX-54) and the European Research Council (ERC, PlastInhib). X.Y. is supported by the China Scholarship Council (CSC).

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Authors and Affiliations

  1. Institute of Biology of the École Normale Supérieure (IBENS), CNRS, Inserm, PSL Research University, Paris, France

    Xiaojuan Yang & Christian G. Specht

  2. East China Normal University, Shanghai, China

    Xiaojuan Yang

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  1. Xiaojuan Yang
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  2. Christian G. Specht
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Correspondence to Christian G. Specht .

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Editors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

    Nobuhiko Yamamoto

  2. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan

    Yasushi Okada

Appendix: Imaging Buffers

Appendix: Imaging Buffers

Gloxy buffer: 0.5 mg/ml glucose oxidase, 40 μg/ml catalase, 0.5 M d-glucose, 50 mM β-mercaptoethylamine (MEA), in PBS pH 7.4, degassed with N2 [3].

OxEA buffer: 3% (v/v) oxyrase (EC-Oxyrase, Sigma-Aldrich), 1 M sodium dl-lactate, 50 mM MEA, in PBS pH 8.0 [67].

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Yang, X., Specht, C.G. (2020). Practical Guidelines for Two-Color SMLM of Synaptic Proteins in Cultured Neurons. In: Yamamoto, N., Okada, Y. (eds) Single Molecule Microscopy in Neurobiology . Neuromethods, vol 154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0532-5_9

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  • DOI: https://doi.org/10.1007/978-1-0716-0532-5_9

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