Electron tomography of immunolabeled cryosections

Electron tomography (ET) and immunolabeling of cells and tissues are both established techniques that give the cell biologist important insights into structure function relationships. While immunolabeling provides localization of specific macromolecules to a structure of interest, ET provides a high-resolution, three-dimensional (3D) perspective that allows one to characterize fine-structural detail and connectivity between compartments that often cannot be seen by other approaches. Combining the two techniques by means of preparing tomographic reconstructions of immunolabeled samples results in a powerful method for localizing macromolecules within the 3D context of a cell. Several different immunolabeling techniques can provide samples that are suitable for tomography. Most of these techniques favor certain structures or classes of antibodies. Here we describe a method by which the Golgi complex and other organelles in mammalian cells are immunolabeled with the technique pioneered by Tokuyasu, and subsequently analyzed by ET. The data generated by this method not only gives a 3D perspective on the labeled structures, they can also be compared directly with similar samples, prepared by rapid-freezing and freeze-substitution, to give a broader, high-resolution view of the cell.