A novel 3D immuno-electron microscopy and its application to the Golgi apparatus.

Serial section scanning electron microscopy (SEM) is useful for revealing the three-dimensional (3D) architecture of organelles by acquiring backscattered electron images of ultrathin serial sections of resin-embedded tissues on solid substrates. However, comprehensive analyses of organelle function require a combination of ultrastructural and molecular localization data. In the present study, we developed a novel 3D immuno-electron microscopy (immuno-EM) approach that combines Tokuyasu cryosectioning with serial section SEM to elucidate the spatial distribution of organelle-associated proteins. Thick cryosections of tissues were immunolabeled with primary antibodies and FluoroNanogold-conjugated secondary antibodies, followed by gold enhancement, resin embedding, and serial sectioning and SEM. Serial tomographic images of organelles were aligned and segmented to generate 3D reconstructions. To demonstrate the effectiveness of the method, we visualized the localization of GM130, a representative cis-Golgi matrix protein, in a 3D model of the Golgi apparatus in rat pituitary gonadotropes. The 3D model revealed a spherical Golgi apparatus composed of five cisternae arranged in cis-trans order, with GM130 localized on the outer cisternae, consistent with previous findings. Our 3D immuno-EM technique enables the detailed 3D visualization of the Golgi apparatus and other organelles as well as analyses of the spatial distribution of target proteins in their 3D reconstructions.