Biochemical, electron microscopic and immunohistological observations of cationic detergent-extracted cells: detection and improved preservation of microextensions and ultramicroextensions

We demonstrate in biochemical experiments that cultured cells needed to be kept in 4% formaldehyde for at least 60 min at room temperature or for 20 min at 37°C to irreversibly crosslink most of the polypeptides. Also, fragmentation of fragile microextensions was observed after Triton X-100 extraction depending on concentration and extent of crosslinking. We also report on a novel fixation procedure that includes the cationic detergent dodecyltrimethylammonium chloride (DOTMAC). Treatment of NIH3T3 cells with DOTMAC resulted in complete removal of membrane lipids and in good preservation of the cytoskeleton in microextensions as well as preservation of ultramicroextensions of <0.05μm in diameter that have not been observed previously unless glutaraldehyde was used. Stress fibers and microextensions of DOTMAC-extracted cells were readily stained with anti-β-actin antibodies, and antibodies to vinculin and moesin stained focal contacts and microextensions, respectively.Some microextensions were fragmented by the standard Triton X-100 permeabilization method. By contrast, DOTMAC completely extracted membrane lipids while maintaining the cytoskeleton of microextensions. Thus, DOTMAC treatment may provide a valuable new tool for the reliable visualization of previously undetectable or poorly detectable antigens while preserving the actin cytoskeleton of microextensions.Cellular events are based on specific inter- and intra-molecular interactions of many molecules. To experimentally dissect such interactions, it is important to maintain as closely as possible the cytosolic environment of the living cell. Detergents are frequently employed to disrupt cells for the demonstration and isolation of functionally relevant protein complexes and/or to selectively extract certain proteins. For example, the non-ionic detergents Triton X-100, Tween 20 or Brij, have been widely used for histochemical purposes or in biochemical experiments to separate detergent-soluble from a detergent