Johnson A B, Bettica A
Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461.
Am J Anat. 1989 Jun-Jul;185(2-3):335-41. doi: 10.1002/aja.1001850228.
On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.
对中间丝等结构进行网格上的免疫金标记一直难以实现。据推测，这是因为这些结构比超薄切片本身更细，而且金标记试剂会留在表面，无法穿透环氧树脂。然而，许多病理组织和其他组织主要是以环氧树脂包埋块的形式存在，因此能够检测此类细微结构的包埋后金标程序将很有用。本研究旨在调查一名患有亚历山大病的儿童经戊二醛固定、环氧树脂包埋的脑活检组织中的星形胶质细胞中间丝抗原胶质纤维酸性蛋白（GFAP）。开发了一种用于进行网格上免疫金标记的方案，该方案可将金试剂的非特异性沉积降至最低。该方法在金试剂的洗涤液和稀释液中使用了卵清蛋白和脱脂牛奶，在抗体和正常血清的稀释液中使用了添加吐温-20和高氯化钠的相同溶液。在用偏高碘酸盐和过氧化氢蚀刻的网格中，星形胶质细胞中间丝仅偶尔被稀疏标记。然而，当采用乙醇钠蚀刻程序时，星形胶质细胞中间丝上获得了广泛的标记。相比之下，亚历山大病特有的较大的病理性罗森塔尔纤维在两种蚀刻程序后均被标记，但乙醇蚀刻后标记增强。后固定材料的标记要少得多。这些发现表明，包埋后程序可用于环氧树脂包埋材料，以免疫标记中间丝等细微结构。
