Immunogold Labeling of Cultured Cells and Virus Particles for Electron Microscopy and Cryo-Electron Microscopy Applications

Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) were first realized in the early 1980s [1] and mid-1990s [2, 3]. Since then both methods have quickly become a powerful tools in structural biology. The major advantage is that they allow for the acquisition of images of biological specimens persevered in their native state after they are plunge-frozen onto carbon-coated grids. When a high voltage transmission electron microscope is used to image the specimen, the resolution reaches the molecular level, which permits the visualization of structures that have not been seen with the conventional electron microscopy. During the past thirty years, the focus has been mainly on ultrastructural analysis, with limited success in the development and application of compatible labeling techniques for assessing the localization of macromolecules. One obstacle for immunogold labeling of frozen-hydrated specimens is the maintenance of specimen viability and structural integrity during the process of immunogold labeling. Most current immunogold procedures require gentle chemical fixation followed by several incubations in buffered solutions. Application of these procedures potentially alters specimen structure.