Detection of epidermal growth factor receptor by scanning electron microscopy

Abstract

A method of immunocytochemistry and low-voltage scanning electron microscopy (SEM) is described for visualization of the epidermal growth factor membrane receptor (EGFR). The specific labelling is achieved of antigenic sites on the surface of prefixed cells. The advantage of this approach over existing techniques is the capability for unlimited high-resolution surface examination at the single cell level. This is achieved by using low acceleration voltage (V0) and either very thin or no coating of the specimens to prevent the label from being masked. Furthermore, by using conventional field emission SEM and a highly sensitive detector for backscattered electrons, detection of the gold-conjugate (< 10 nm in diameter) becomes possible even at low V0. A431 cells (human epidermoid carcinoma) show intercellular variability in their EGFR area density. Highest density was recorded upon cells in the mitotic stage of the cell cycle due to a decrease in the relative surface of rounded versus flattened cells. At the ultrastructural level a marked heterogeneity was also seen on the surface of contracted cells, where enhanced labelling could be observed only at the tips of microvilli. In contrast, spread cells displayed a homogeneous receptor distribution due to their smooth surface.