Stump R F, Pfeiffer J R, Seagrave J, Oliver J M
University of New Mexico School of Medicine, Department of Pathology, Albuquerque 87131.
J Histochem Cytochem. 1988 May;36(5):493-502. doi: 10.1177/36.5.2965720.
Immunogold labeling and silver enhancement techniques are widely used to determine density and distribution of cell membrane receptors by light and transmission electron microscopy. However, these techniques have not been widely used for receptor detection by scanning electron microscopy. We used antigen- or protein A-conjugated colloidal gold particles, together with silver enhancement, sequential secondary and back-scattered electron imaging (SEI and BEI), and digital image processing, to explore cell surface distribution of IgE-receptor complexes on RBL-2H3 cells, a rat leukemia line that provides a model for the study of mucosal mast cells. Cells were first incubated with a monoclonal antidinitrophenol IgE (anti-DNP-IgE) that binds with high affinity to cell surface IgE receptors. The resulting IgE-receptor complexes were cross-linked either with the multivalent antigen, DNP-BSA-gold, or with a polyclonal anti-IgE antibody. Antibody-treated cells were labeled after fixation with protein A-gold. Fixed, gold-labeled cell monolayers were silver enhanced (or not), dehydrated, critical point-dried, and coated with gold-palladium (for SEI analysis) or carbon (for combined SEI/BEI analysis). They were observed in an Hitachi S800 SEM equipped with a field emission tip and a Robinson backscattered electron detector. An image processor (MegaVision 1024XM) digitized images directly from the S800 microscope at 500-1000 line resolution. Silver enhancement significantly improves detection of gold particles in both SEI and BEI modes of SEM. On gold-palladium-coated samples, 20-nm particles are resolved by SEI after enhancement. BEI resolves 15-nm particles without enhancement and 5- or 10-nm particles are resolved by BEI on silver-enhanced, carbon-coated samples. Neither BEI nor SEI alone can yield high resolution topographical maps of receptor distribution (BEI forms images on the basis of atomic number contrast which reveals gold but not surface features). Image analysis techniques were therefore introduced to digitize, enhance, and process BEI and SEI images of the same field of view. The resulting high-contrast, high-resolution images were superimposed, yielding well-resolved maps of the distribution of antigen-IgE-receptor complexes on the surface of RBL-2H3 mast cells. The maps are stored in digital form, as required for computer-based quantitative morphometric analyses. These techniques of silver enhancement, combined BEI/SEI imaging, and digital image analysis can be applied to analyze density and distribution of any gold-labeled ligand on its target cell.
免疫金标记和银增强技术被广泛用于通过光学显微镜和透射电子显微镜来确定细胞膜受体的密度和分布。然而，这些技术尚未广泛用于通过扫描电子显微镜进行受体检测。我们使用抗原或蛋白A偶联的胶体金颗粒，结合银增强、连续二次电子和背散射电子成像（SEI和BEI）以及数字图像处理，来探索RBL-2H3细胞（一种大鼠白血病细胞系，为黏膜肥大细胞的研究提供了模型）表面IgE受体复合物的分布。细胞首先与单克隆抗二硝基苯酚IgE（抗DNP-IgE）孵育，该抗体与细胞表面IgE受体具有高亲和力结合。产生的IgE受体复合物用多价抗原DNP-BSA-金或多克隆抗IgE抗体交联。用蛋白A-金固定后对抗体处理的细胞进行标记。固定的、金标记的细胞单层进行银增强（或不增强）、脱水、临界点干燥，然后涂覆金钯（用于SEI分析）或碳（用于联合SEI/BEI分析）。在配备场发射尖端和罗宾逊背散射电子探测器的日立S800扫描电子显微镜中观察它们。图像处理器（MegaVision 1024XM）以500-1000线分辨率直接从S800显微镜数字化图像。银增强显著提高了扫描电子显微镜SEI和BEI模式下金颗粒的检测。在涂覆金钯的样品上，增强后通过SEI可分辨20纳米颗粒。BEI在未增强时可分辨15纳米颗粒，在银增强、涂覆碳的样品上通过BEI可分辨5或10纳米颗粒。单独的BEI和SEI都不能产生受体分布的高分辨率地形图（BEI基于原子序数对比度形成图像，可显示金但不能显示表面特征）。因此引入图像分析技术来数字化、增强和处理同一视野的BEI和SEI图像。将得到的高对比度、高分辨率图像叠加，生成RBL-2H3肥大细胞表面抗原-IgE-受体复合物分布的清晰地形图。这些地形图以数字形式存储，这是基于计算机的定量形态分析所需要的。银增强、联合BEI/SEI成像和数字图像分析这些技术可用于分析任何金标记配体在其靶细胞上的密度和分布。
