Taguchi Kensei, Elias Bertha C, Qian Subo, Brooks Craig R
Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, United States.
Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, United States; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, United States.
Methods Cell Biol. 2019;153:231-253. doi: 10.1016/bs.mcb.2019.05.004. Epub 2019 Jun 25.
Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.
据估计，肾病影响着全球15%的人口。自噬是真核细胞中一条关键的稳态途径，它与多种病理状态有关。在肾脏中，自噬已被证明可调节急性和慢性损伤。尽管自噬在肾病中很重要，但目前可用的精确监测肾组织中自噬通量的技术很少。在此，我们描述了一种使用RFP-GFP-LC3报告基因小鼠和超分辨率显微镜来量化自噬通量的改进技术。利用结构光照明显微镜，我们可以分辨肾小管细胞内的单个自噬体。我们描述了载玻片的制备、染色、成像和数据处理。利用三维表面渲染，根据数量、大小、荧光和缺血后的自噬通量对自噬体进行分类和量化。
