Subcellular localization of immune mediators and other proteins

While conventional transmission electron microscopy (TEM) has been used to unveil ultrastructural features of eosinophils, such as distinct secretory patterns, TEM combined with molecular detection methods has been applied for precise localization of proteins to intracellular compartments. Visualization of specific proteins at the nanoscale level is achieved with immunogold EM, a technique that investigates the protein of interest by performing immunolabeling with a primary antibody against the target molecule followed by a secondary antibody (against primary antibody) conjugated with gold nanoparticles with sufficient contrast to be observed by TEM. We have mostly been applying pre-embedding immunonanogold EM, which means antigen labeling before all regular procedures for TEM, thus enabling improved antigen preservation. In this chapter, we address the subcellular localization of different types of immune mediators including cytokines, cationic proteins, and tetraspanins, and other proteins in human eosinophils during resting conditions and immune responses. We also show representative quantitative analyses that provided additional information on the pattern, frequency, and level of the immunolabeling. Application of immunonanogold EM has helped to appreciate the complex collection of biomolecules, which are constitutively expressed in human eosinophils as well as the secretory pathways involved in the release of these mediators during eosinophil inflammatory responses.