A novel permeabilization protocol to obtain intracellular 3D immunolabeling for electron tomography

Electron tomography (ET) is a very important high-resolution tool for 3D imaging in cell biology. By combining the technique with immunolabeling, ET can provide essential insights into both cellular architecture and dynamics. We recently developed a protocol to achieve 3D immunolabeling of intracellular antigens without the need for uncontrolled permeabilization steps that cause random, extensive cell membrane disruption. Here we describe this novel method based on well-controlled permeabilization by targeted laser cell perforation. Mechanical permeabilization of the plasma membrane can be applied at specific sites without affecting other parts of the plasma membrane and intracellular membranes. Despite the relatively small opening created in the plasma membrane, the method allows specific 3D immunolocalization of cytoplasmic antigens in cultured cells by a pre-embedment protocol. The approach is unique and leads to a superior ultrastructural preservation for transmission electron microscopy and electron tomography.