freeze-fracture, cytochemistry, immunolabeling, lipopolysaccaride
The freeze-fracture technique provides large and detailed ultrastructural views of cellular membranes in the transmission electron microscope. The major goal of freeze-fracture cytochemistry is the identification and localization of membrane components such as proteins or lipids for studying their distribution, dynamics or their relationship to specialized membrane domains. The most versatile and effective technique in freeze-fracture cytochemistry is termed freeze-fracture replica immunolabeling (FRIL). Using this technique chemically unfixed cellular samples are rapidly cryofixed in their natural habitat. They are fractured, replicated and immobilized by platinum-carbon evaporation followed by a careful sodium dodecyl sulfate (SDS) treatment. The detergent SDS removes most of the cellular material except those molecules that are in direct contact to the platinum/carbon replica. Immunogold labeling of these replica-bound biomolecules visualizes their spatial organization in high resolution on planar views of cellular membranes.