Immuno-localization of ESCRT Proteins in Virus-Infected Cells by Fluorescence and Electron Microscopy

Methods Mol Biol. 2019:1998:73-92. doi: 10.1007/978-1-4939-9492-2_6.

Abstract

Many enveloped viruses utilize the cellular ESCRT pathway for budding, even flaviviruses, which form viral particles inside replication organelles derived from the endoplasmic reticulum (ER). In this section, we introduce methods for detecting several ESCRT subunit proteins in virus-infected cells by immunofluorescence microscopy and immunoelectron microscopy (immuno-EM). We also introduce a new method; correlative light microscopy and electron microscopy (CLEM), which allows the observation of target structures with both high-resolution EM and fluorescence labeling.

Keywords: Conventional transmission EM (TEM); Correlative light microscopy and electron microscopy (CLEM); Dengue virus (DENV); Fluorescence light microscopy; Immunoelectron microscopy; Japanese encephalitis virus (JEV); Positive strand RNA virus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Cell Culture Techniques / methods
  • Cell Line
  • Dengue Virus / immunology
  • Encephalitis Virus, Japanese / immunology
  • Endoplasmic Reticulum / metabolism
  • Endoplasmic Reticulum / ultrastructure*
  • Endoplasmic Reticulum / virology
  • Endosomal Sorting Complexes Required for Transport / chemistry
  • Endosomal Sorting Complexes Required for Transport / immunology
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Gold / chemistry
  • Humans
  • Mesocricetus
  • Metal Nanoparticles / chemistry
  • Microscopy, Electron, Transmission / methods*
  • Microscopy, Fluorescence / methods
  • Microscopy, Immunoelectron / methods
  • Molecular Imaging / methods*
  • Staining and Labeling / methods
  • Virion / chemistry

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Gold