Heinzmann U, Höfler H
GSF-Forschungszentrum für Umwelt und Gesundheit, GmbH, Institut für Pathologie, Postfach, Germany.
Histochemistry. 1994 Feb;101(2):127-34. doi: 10.1007/BF00269359.
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.
本文描述了一种免疫细胞化学和低电压扫描电子显微镜（SEM）相结合的方法，用于可视化表皮生长因子膜受体（EGFR）。该方法可对固定细胞表面的抗原位点进行特异性标记。与现有技术相比，这种方法的优势在于能够在单细胞水平上进行无限高分辨率的表面检查。这是通过使用低加速电压（V0）以及对标本进行极薄涂层或不涂层处理来防止标记被掩盖而实现的。此外，通过使用传统的场发射扫描电子显微镜和高灵敏度的背散射电子探测器，即使在低V0下也能够检测到直径小于10 nm的金偶联物。A431细胞（人表皮样癌）在其EGFR面积密度上表现出细胞间变异性。由于圆形细胞与扁平细胞相对表面积的减少，在细胞周期的有丝分裂阶段的细胞上记录到最高密度。在超微结构水平上，收缩细胞表面也存在明显的异质性，仅在微绒毛尖端观察到标记增强。相比之下，铺展细胞由于其表面光滑而呈现出均匀的受体分布。
