Cramer E M, Werb Z, Bainton D F
Department of Pathology, University of California, San Francisco 94143-0506.
Histochem J. 1988 Feb;20(2):108-16. doi: 10.1007/BF01746611.
We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5'-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killed Escherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5'-nucleotidase onto the adhering particle was fact or artifact, we localized 5'-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5'-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.
我们使用了以硝酸铅作为捕获剂的酶细胞化学方法以及电子显微镜水平的免疫方法，来定位大鼠腹膜常驻巨噬细胞在吞噬乳胶珠或热灭活大肠杆菌过程中，质膜5'-核苷酸酶在细胞初始相互作用时的位置。在静止的巨噬细胞中，细胞化学反应产物沿质膜外表面均匀分布。然而，当细胞吞噬乳胶珠或细菌时，反应产物覆盖了附着颗粒的整个表面。为了确定5'-核苷酸酶在附着颗粒上的明显重新分布是真实情况还是人为假象，我们使用单克隆抗体和免疫金标记技术对5'-核苷酸酶进行定位。在结合或开始摄取细菌的巨噬细胞中，金颗粒沿质膜分布，但细胞-细菌内化部位除外。更重要的是，附着的细菌没有金颗粒，因此其表面没有5'-核苷酸酶。事实证明乳胶珠不适宜作为测试颗粒，因为金颗粒会非特异性地黏附在它们上面。我们得出结论，磷酸铅反应产物的人为重新分布是酶细胞化学方法用于细胞表面时的一个主要缺点，而免疫金标记技术更可靠。
