Menghi G, Marchetti L, Bondi A M, Accili D, Sabbieti M G, Materazzi G
Department of Comparative Morphological and Biochemical Sciences, University of Camerino, Italy.
Histol Histopathol. 1999 Jul;14(3):687-95. doi: 10.14670/HH-14.687.
In the present study we report the development of an ultrastructural electron microscopic double-sided staining technique that, using gold probes of 10 nm and enhancement of the gold signal by silver amplification, allows the demonstration of two antigenic sites on the same section. The labeling was carried out in the following manner: one face of uncoated floating grids was incubated with an antibody directed to alpha-amylase, followed by a secondary gold-labeled antibody, amplification of gold particles, drying and carbon coating; subsequently, the reverse face of the same grid, was processed for lectin cytochemistry, with and without sialidase digestion, and it was incubated with HRP-conjugated lectins, anti-HRP antibody and protein-A gold. Also the reverse sequence of steps and amplification of gold signal after the first or second labeling were experimented. The resultant small and large particles revealed different distributional patterns of antigenic sites on the opposite faces of the same tissue section. The transparency of the resin-embedded ultrathin sections in the electron beam allowed the simultaneous visualization of the gold probes of different sizes present on the two faces. The analysis of immunolabeling revealed that the alpha-amylase is chiefly secreted by the parotid and submandibular glands. The application of this double-sided staining technique also indicated that, when present in glycosylated form, the alpha-amylase enzyme does not contain sialic acid in the submandibular and sublingual glands; conversely, its location on the electron-dense areas of target granules in the parotid acinar cells seems to suggest that a sialylated isoenzymatic form can occur within these granule regions where sialic, acid linked to beta-galactose, was found to be located.
在本研究中，我们报告了一种超微结构电子显微镜双面染色技术的开发。该技术使用10纳米的金探针，并通过银增强来放大金信号，从而能够在同一切片上显示两个抗原位点。标记过程如下：将未包被的漂浮网格的一面与抗α淀粉酶抗体孵育，接着与二级金标记抗体孵育，放大金颗粒，干燥并进行碳包被；随后，对同一网格的另一面进行凝集素细胞化学处理，包括有无唾液酸酶消化，然后与辣根过氧化物酶（HRP）偶联的凝集素、抗HRP抗体和蛋白A金孵育。还对步骤的反向顺序以及第一次或第二次标记后的金信号放大进行了实验。所得的大小颗粒显示出同一组织切片相对面上抗原位点的不同分布模式。树脂包埋超薄切片在电子束中的透明度使得能够同时观察到两面存在的不同大小的金探针。免疫标记分析表明，α淀粉酶主要由腮腺和颌下腺分泌。这种双面染色技术的应用还表明，当以糖基化形式存在时，颌下腺和舌下腺中的α淀粉酶酶不含唾液酸；相反，它在腮腺腺泡细胞中靶颗粒的电子致密区域的定位似乎表明，在这些发现唾液酸与β-半乳糖相连的颗粒区域内可能存在唾液酸化的同工酶形式。
