Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure
Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure
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DOI：
10.2220/biomedres.41.161
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发表时间：
2020-01-01
影响因子：
1.2
通讯作者：
Watanabe, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Koga, Daisuke;Kusumi, Satoshi;Watanabe, Tsuyoshi
锇浸渍法是扫描电镜观察细胞器三维超微结构的有效方法。在传统的锇浸渍法中，组织在20摄氏度下浸入稀释的四氧化锇溶液中数天，以从细胞的冷冻破裂表面去除可溶性胞质蛋白，该过程的最佳持续时间取决于细胞类型。为了提高锇浸渍过程的效率，我们系统地研究了锇浸渍过程中反应温度与时间的关系。在高于20摄氏度的温度下的治疗大大缩短了所需的时间，以消除细胞溶质蛋白从冷冻破裂的表面的标本与最佳持续时间为锇浸渍的肝细胞在30，40，50和60摄氏度分别为30，15，5和1小时。考虑到浸渍样品的稳定性和重现性，我们认为最合适的温度为30至40 ℃。这种快速的锇浸渍法成功地用于观察小脑浦肯野细胞和肾脏近曲小管细胞的三维超微结构。这种简单和可重复的快速锇浸渍协议应fmd广泛的吸引力在各种细胞类型的细胞器的3D分析。
The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20 degrees C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20 degrees C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60 degrees C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40 degrees C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should fmd wide appeal for the 3D analysis of cellular organelles in various cell types.