Abstract
Three-dimensional (3D) protein localization at the electron microscopic level is of great benefit when determining the precise distribution of a given protein relative to complex subcellular structures. We have developed several 3D ultrastructural labeling approaches for use with higher voltage electron microscopy. High voltage electron microscopy allows for the use of much thicker sections than conventional transmission electron microscopy. Many of our staining protocols are based on the photooxidation of diaminobenzidine (DAB) by fluorescent markers. As a fluorescent dye is excited in the presence of DAB, the reactive oxygen generated gradually oxidizes DAB into an insoluble polymer which can be rendered electron dense. We previously demonstrated that photooxidation by the fluorophore eosin can be used for high resolution immunolocalization. Because of its small size, eosin-conjugated reagents penetrate well into tissue slices, providing labeling through a reasonable depth of tissue. When combined with high voltage electron microscopy using thick sections, the 3D distribution of a variety of proteins can be visualized. Analysis and interpretation of these 3D labeling patterns is further facilitated by the use of electron tomography, which allows for the derivation of 3D structure from a series of images taken at different orientations. In this review, several examples of 3D protein localization using these technologies are presented.
