Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.
Immunolabeling of adenohypophysial cells with protein A-colloidal gold--antibody complex for electron microscopy: use of the freeze-substitution technique in tissue preparation.
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用于电子显微镜的用蛋白 A-胶体金-抗体复合物对腺垂体细胞进行免疫标记：在组织制备中使用冷冻替代技术。
DOI：
10.1177/32.7.6736623
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
S. Daikoku
中科院分区：
文献类型：
--
作者：
S. Hisano;T. Adachi;S. Daikoku
通过比较冷冻替代法和传统方法制备的垂体前叶细胞，研究了冷冻替代法制备组织用于电镜免疫组织化学的价值。对包埋在Epon中的组织的超薄切片进行免疫染色。用蛋白A-胶体金-抗体（PAG-Ab）复合物双标技术，在同一切片上显示促肾上腺皮质激素（ACTH）和催乳素（PRL）抗原。细胞超微结构的保存是上级在FS获得的制剂。分泌颗粒上可见金标记，促肾上腺皮质激素细胞中也可见金标记。从金颗粒的数量来看，FS获得的制剂中的标记更强烈。在双标记过程中，其中PA-小胶体金-抗-PRL复合物和PA-大胶体金-抗-ACTH复合物依次应用于切片，未观察到与小和大金颗粒的交叉标记。它的结论是，如果抗血清是足够的特异性，FS和pAG-Ab复合物的使用是非常有效的肽免疫组化。然而，在双标记过程中，必须通过使用过量的抗体使pAG的Fc结合位点饱和。
The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.