The Hormel Institute, University of Minnesota, Austin, MN, USA.
Division of Gynecologic Oncology, Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, CA, USA.
Methods Mol Biol. 2021;2255:135-147. doi: 10.1007/978-1-0716-1162-3_12.
Transmission electron microscopy (TEM) is an all-in-one tool to visualize the complex systems of any specimen that is 1 nm in size or smaller. The current chapter provides detailed guidelines for imaging morphological changes during programmed cell necrosis using TEM as a single-step methodology. In this protocol, a novel aldehyde dehydrogenase inhibitor is used to induce cell programmed necrosis in ovarian cancer cell lines (A2780 and SKOV3). This process is followed by gradient dehydration with ethanol, chemical fixation, sampled grid preparation, and staining with 0.75% uranyl formate. Following fixation and grid preparation, cells are imaged using TEM. The resulting images reveal morphological changes consistent with necrotic morphology, including swelling of cells and organelles, appearance of vacuoles, and plasma membrane rupture followed by leakage of cellular contents. The current approach allows a single-step methodology for characterization of cell-programmed necrosis in cells based on morphology.
透射电子显微镜（TEM）是一种将大小为 1nm 或更小的任何标本的复杂系统可视化的综合工具。本章提供了使用 TEM 作为单一方法来观察程序性细胞坏死过程中形态变化的详细指南。在该方案中，使用新型醛脱氢酶抑制剂诱导卵巢癌细胞系（A2780 和 SKOV3）发生细胞程序性坏死。接下来进行梯度乙醇脱水、化学固定、取样网格制备以及 0.75%的甲酸乌来霉素染色。固定和网格制备后，使用 TEM 对细胞进行成像。所得图像显示出与坏死形态一致的形态变化，包括细胞和细胞器肿胀、出现空泡以及细胞膜破裂后细胞内容物泄漏。目前的方法允许基于形态学对细胞程序性坏死进行单一方法学的特征描述。
