TEM Embedding Technique for Immunocytochemistry of Cultured Cells

Abstract

Vero cells (African green monkey kidney epithelium) contain very dense cytoplasm with numerous ribosomes and mitochondria. They also contain an impressive network of cytoskeletal proteins, especially vimentin intermediate filament. Vimentin is present in all cells of mesenchymal origin, but their localization in the cell is usually restricted to a perinuclear position. Vero cells exhibit large bundles of vimentin filaments in a network, coursing throughout the cell. Additionally, many vimentin filaments connect to each other in an intermediate filament focus, or vertex. We investigated these vimentin vertices using immunofluorescence, scanning and transmission electron microscopy.

The processing of cultured cells for transmission electron microscopy is a difficult task, especially when one desires to preserve in situ relationships. When sectioning epithelial cell lines, a single cell layer (approximately three micrometers) of tissue is available, so a few thick sections will remove all the cells in the block. A traditional method for TEM of cultured cells is to disrupt these cells from their substrate and then centrifuge these cells into a pellet which can be sectioned. While it may be easier to obtain thin sections of cultured cells employing this method, many characteristics of the cells cannot be adequately investigated, including cell-to-cell junctions, cell shape on a substrate, cell morphology associated with a substrate, position of organelles in a cell growing on a substrate, and morphology and relationship of mitotic cells.

Growing cells on Thermanox cover slips and embedding in LR White polymerized in gelatin capsules at 55°C affords an effective method of viewing and labeling cells in culture. When the resin has been polymerized, the Thermanox coverslip is carefully removed from the block by using a razor blade to pry it off. The cells remain in the LR White, exposing them to the surface of the block. in fact, the very first sections of the block contain cells. Although this prevents one from facing the block, the exposed surface is usually flat enough to obtain good thin sections.