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Using Sequential Dual-Immunogold-Silver Labeling and Electron Microscopy to Determine the Fate of Internalized G-Protein-Coupled Receptors Following Agonist Treatment
Book chapter   Peer reviewed

Using Sequential Dual-Immunogold-Silver Labeling and Electron Microscopy to Determine the Fate of Internalized G-Protein-Coupled Receptors Following Agonist Treatment

Elisabeth J. Van Bockstaele, Janet L. Kravets, Xin-Mei Wen and Beverly A. S. Reyes
Transmission Electron Microscopy Methods for Understanding the Brain, pp 139-166
20 Apr 2016
DOI: https://doi.org/10.1007/7657_2015_77

Abstract

Endosomes Interacting proteins Receptor internalization Trafficking Ultrastructure
Internalization of ligand-receptor complexes is a consequence of receptor signaling resulting in receptors being recycled to the cell surface or transported to lysosomes for eventual degradation. Resolving the fate of internalized receptors allows predictions to be made regarding changes in cellular sensitivity and the ability to test specific synaptic models of interaction between related receptor systems. Dual-labeling immunohistochemistry employing visually distinct immunoperoxidase and immunogold markers has been an effective approach for elucidating complex receptor profiles at the synapse and to definitively establish the localization of individual receptors and ligands to common cellular profiles. However, the combination of dual-immunogold-silver labeling of distinct antigens offers some unique benefits for resolving interactions between G-protein-coupled receptors (GPCR), their interacting proteins, or downstream effectors. This approach provides superior subcellular localization of the antigen of interest while preserving optimal ultrastructural morphology. Pre-embedding methods are also more appropriate than post-embedding methods for localization of immunoreactivity at extrasynaptic sites making quantification of GPCR distribution more suitable. Here, we provide detailed methodologies of the use of different sized immunogold particles to analyze the association of the mu-opioid receptor (MOR) with lysosome-associated membrane protein (LAMP), an integral membrane protein that is associated with lysosomes or early endosome antigen (EEA), a protein associated with early endosomes, following intracerebroventricular (icv) administration of saline or the opiate agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) in the striatum of male Sprague-Dawley rats.

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