DETECTION OF EPIDERMAL GROWTH-FACTOR RECEPTOR BY SCANNING ELECTRON-MICROSCOPY

A method of immunocytochemistry and low-voltage scanning electron micr oscopy (SEM) is described for visualization of the epidermal growth fa ctor membrane receptor (EGFR). The specific labelling is achieved of a ntigenic sites on the surface of prefixed cells. The advantage of this approach over existing techniques is the capability for unlimited hig h-resolution surface examination at the single cell level. This is ach ieved by using low acceleration voltage (V-0) and either very thin or no coating of the specimens to prevent the label from being masked. Fu rthermore, by using conventional field emission SEM and a highly sensi tive detector for backscattered electrons, detection of the gold-conju gate (<10 nm in diameter) becomes possible even at low V-0. A431 cells (human epidermoid carcinoma) show intercellular variability in their EGFR area density. Highest density was recorded upon cells in the mito tic stage of the cell cycle due to a decrease in the relative surface of rounded versus flattened cells. At the ultrastructural level a mark ed heterogeneity was also seen on the surface of contracted cells, whe re enhanced labelling could be observed only at the tips of microvilli . In contrast, spread cells displayed a homogeneous receptor distribut ion due to their smooth surface.