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Proximity ligation assay (PLA) is a newly developed technique that outperforms the traditional immunoassays for visualizing the in situ endogenous protein-protein interactions and localizations and the activation of proteins in cell culture systems as well as in tissue sections. PLA, when combined with cellular marker staining, becomes a powerful approach to identify differential interaction of the proteins of endosomal sorting complex required for transport (ESCRT) at distinct stages of virus infection. In this chapter, we describe a PLA protocol to study the localization and interaction between the ESCRT protein TSG101 and endosomal markers in early stages of viral endocytosis in in vitro infected cells.

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.

Most endosomal sorting complex required for transport (ESCRT)-III proteins are not fully functional when expressed as fusion of fluorescent or epitope tags, frequently making the use of specific antibodies the only available method for their detection. Heterologous expression of ESCRT-III proteins in bacteria often results in the formation of insoluble aggregates or inclusion bodies that interfere with their purification. However, inclusion bodies are usually pure protein aggregates with high antigenicity. In addition, since proteins within inclusion bodies are presented in a range of folding states, immunization with inclusion bodies can potentially result in antibodies with specificity for different folding states of the protein under study. We describe here a protocol to isolate bacterial inclusion bodies of plant ESCRT-III proteins for production of polyclonal antibodies. We also describe a nitrocellulose-based immunoaffinity purification method that allows the immobilization of ESCRT-III proteins and the subsequent isolation of specific antibodies from a crude serum.