Immunoelectron Microscopic Localization of Intermediate Filament Vimentin in Neuroblastoma Cells: Methods

Abstract

The cytoskeleton of many cell types contains three prominent filamentous elements: intermediate filaments; actin-containing microfilaments and microtubules. Five antigenically distinct classes of intermediate filaments (IFs) have been identified on the basis of their protein subunit composition: keratin; desmin; glial fibrillary acidic protein; neurofilament triplet protein and vimentin. Although substantial progress in identifying and determining the functional roles of these filamentous proteins has been made with immunof1uorescent techniques, relatively little has been accomplished with immunoelectron microscopy. Previously, it has been difficult to simultaneously preserve ultrastructural detail, maintain antigenicity and ensure antibody penetration into dense cytoskeletal structures. The non-ionic detergent, Triton X-100 has been used, prior to or after fixation, to facilitate macromolecular access to deeply localized antigens. This extraction procedure maintains antigenicity as mirrored by immunofluorescence microscopy. The present study presents a reliable method that preserves dense filamentous cellular organelles and permits immunocytochemica1 localization of vimentin in cultured neuroblastoma cells.