Kusumi Satoshi, Koga Daisuke, Watanabe Tsuyoshi, Shibata Masahiro
Division of Morphological Sciences, Kagoshima University Graduate School of Medicine and Dental Sciences.
Department of Microscopic Anatomy and Cell Biology, Asahikawa Medical University.
Biomed Res. 2018;39(1):21-25. doi: 10.2220/biomedres.39.21.
We describe a novel immuno-scanning electron microscopy (SEM) technique that combines both Tokuyasu's cryosectioning and section SEM methods. In this technique, semithin cryosections, cut according to the Tokuyasu method, were adhered to glass microscope slides, immunostained for bio-molecules of interest and observed by confocal laser scanning microscopy. The same sections were subsequently embedded in epoxy resin and ultrathin sections were cut on an ultramicrotome. These were then observed by SEM using a backscattered electron detector. Correlation between immunofluorescence and SEM images was performed in the same area of the cryosection. Immuno-SEM was also performed using a FluoroNanogold-labeled secondary antibody. This novel immuno-SEM method can provide ultrastructural information of cell organelles in relation to associated molecules, such as Golgi- and ER-associated proteins. This novel immuno-SEM technique has the potential to be widely used.
我们描述了一种新型免疫扫描电子显微镜（SEM）技术，该技术结合了德久保的冷冻切片法和切片扫描电子显微镜法。在这项技术中，按照德久保方法切割的半薄冷冻切片被粘附到玻璃显微镜载玻片上，针对感兴趣的生物分子进行免疫染色，然后通过共聚焦激光扫描显微镜观察。随后将相同的切片嵌入环氧树脂中，并在超薄切片机上切割超薄切片。然后使用背散射电子探测器通过扫描电子显微镜对这些切片进行观察。在冷冻切片的同一区域进行免疫荧光和扫描电子显微镜图像的相关性分析。还使用氟纳米金标记的二抗进行免疫扫描电子显微镜分析。这种新型免疫扫描电子显微镜方法可以提供与相关分子（如高尔基体和内质网相关蛋白）相关的细胞器超微结构信息。这种新型免疫扫描电子显微镜技术具有广泛应用的潜力。
