Membrane topology information and views of membrane-embedded protein complexes promote our understanding of membrane organization and cell biological function involving membrane compartments. Freeze-fracturing of biological membranes offers both stunning views onto integral membrane proteins and perpendicular views over wide areas of the membrane at electron microscopical resolution. This information is directly assessable for 3D analyses and quantitative analyses of the distribution of components within the membrane if it were possible to specifically detect the components of interest in the membranes. Freeze-fracture replica immunolabeling (FRIL) achieves just that. In addition, FRIL preserves antigens in their genuine cellular context free of artifacts of chemical fixation, as FRIL uses chemically unfixed cellular samples that are rapidly cryofixed. In principle, the method is not limited to integral proteins spanning the membrane. Theoretically, all membrane components should be addressable as long as they are antigenic, embedded into at least one membrane leaflet, and accessible for immunolabeling from either the intracellular or the extracellular side. Consistently, integral proteins spanning both leaflets and only partially inserted membrane proteins have been successfully identified and studied for their molecular organization and distribution in the membrane and/or in relationship to specialized membrane domains. Here we describe the freeze-fracturing of both cultured cells and tissues and the sample preparations that allowed for a successful immunogold-labeling of caveolin1 and caveolin3 or even for double-immunolabelings of caveolins with members of the syndapin family of membrane-associating and -shaping BAR domain proteins as well as with cavin 1. For this purpose samples are cryopreserved, fractured, and replicated. We also describe how the obtained stabilized membrane fractures are then cleaned to remove all loosely attached material and immunogold labeled to finally be viewed by transmission electron microscopy.
Keywords: Caveolae; Caveolar invaginations; Caveolin; Freeze-fracture; Immunogold labeling; Membrane proteins; Membrane topology; Membrane-associated proteins; Nanodomains; Syndapin.