Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Laboratory of Morphology and Image Analysis, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Sci Rep. 2020 Jul 9;10(1):11314. doi: 10.1038/s41598-020-68191-z.
Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.
锇酸后固定和环氧树脂包埋是强有力的和必不可少的处理方法，用于在电子显微镜分析中保存和可视化细胞内的膜结构。然而，这些处理方法会显著降低细胞中大多数荧光蛋白的荧光强度，这为环氧树脂包埋细胞的在树脂中相关的光电子显微镜（CLEM）分析带来了障碍。在本研究中，我们使用了一种远红色荧光蛋白，它在锇染色和环氧树脂包埋后仍保留荧光，从而对环氧树脂包埋的样品进行在树脂中 CLEM。该蛋白的荧光在经 2.5%戊二醛固定和 1%锇酸后固定的环氧树脂包埋样品的细胞 100nm 薄片中被检测到。我们使用定位于线粒体的荧光蛋白对环氧树脂包埋细胞中的线粒体进行了在树脂中 CLEM。使用这种蛋白，我们在环氧树脂包埋样品的细胞薄片中实现了高尔基体和内质网的在树脂中 CLEM。据我们所知，这是第一个报道的远红色荧光蛋白在锇染色和环氧树脂包埋后仍保留其荧光的例子，也是第一个在环氧树脂包埋样品中实现高尔基体和内质网的在树脂中 CLEM 的例子。
