Flatten-peeled: a new approach to freeze-fracture morphology

A new way to obtain in a replica vast pictures of membranes situated at the same level in the preparation is described. The tissue specimen is flattened during fixation with a given orientation. It can be fractured afterwards, either along the plane of fixation or, after rotation, perpendicular to this plane. The method utilizes standard freeze-fracture equipment and readily available materials. This approach has a large range of potential applications, especially with tissues displaying a layered organization such as epithelia, nervous system, etc. It was found very appropriate for studying pre- and postsynaptic membranes in the Torpedo electric organ, to reveal specific junctions between cells in organotypic cultures, and to examine photoreceptors and other layers of the mammalian retina. In those tissues we obtained in a fairly reproducible manner freeze fractures at the desired level and orientation. With Torpedo synapses, vast pictures of the nerve terminals were performed in a plane quasi-parallel to the membrane postsynaptic cells (electroplates). Using double or triple flatten-peeled, it has also been possible to render the whole tissue specimen thin enough to perform gold label fracture [Pinto da Silva and Kan (1984), J. Cell Biol., 99:1156-1161].