Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe.
Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe.
复制标题
使用带有银增强金探针的冷冻固定和冷冻替代皮肤，通过电子显微镜演示桥粒抗原。
DOI：
10.1177/42.5.8157937
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
T. Nishikawa
中科院分区：
文献类型：
--
作者：
H. Shimizu;T. Masunaga;A. Ishiko;T. Hashimoto;D. Garrod;H. Shida;T. Nishikawa
在之前的包埋后免疫金电子显微镜（EM）研究中，各种桥粒抗原的定位在高倍率下是可能的，但在低倍率下则不行。我们开发了一种在低功率和高功率 EM 放大倍数下同时演示表皮桥粒抗原的方法，该方法基于冷冻固定和丙酮冷冻替代，并使用 1 nm 金探针和银增强。将 Lowicryl K11M 的超薄切片与抗桥粒斑蛋白、桥粒胶蛋白或桥粒芯糖蛋白的一抗一起孵育，然后与 1 nm 金缀合的二抗一起孵育。银增强 12 分钟为低功率可视化提供了理想的标记尺寸，而银增强 4-6 分钟则为高功率 EM 观察提供了理想的选择。即使在非常低的放大倍数下，用金探针免疫标记的每个桥粒也被清晰地展示出来。可以轻松确定背景标记的水平，并准确选择高倍观察的感兴趣区域。高倍观察精确展示了桥粒分子精细的超微结构外观。该系统应可用于各种桥粒抗原以及其他感兴趣分子的免疫细胞化学研究。
In a previous post-embedding immunogold electron microscopic (EM) studies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications by a method based on cryofixation and acetone cryosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowicryl K11M were incubated with primary antibodies against desmoplakin, desmocollin, or desmoglein, followed by 1-nm gold-conjugated secondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal for high-power EM observation. Each desmosome immunolabeled with the gold probe was clearly demonstrated, even at very low-power magnification. The level of background labeling could be determined easily and the area of interest for high-power observation selected accurately. The fine ultrastructural appearance of desmosomal molecules was precisely demonstrated on high-power observation. This system should be useful for the immunocytochemical study of a variety of desmosomal antigens as well as other molecules of interest.