Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis
Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis
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DOI：
10.14814/phy2.12306
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发表时间：
2015-02-01
影响因子：
2.5
通讯作者：
El-Achkar, Tarek M.
中科院分区：
文献类型：
--
作者：
Micanovic, Radmila;Khan, Shehnaz;El-Achkar, Tarek M.
激光显微解剖(LMD)是一种非常有用的工具，它可以从组织样本中分离出有限的区域，用于RNA和蛋白质的下游分析。虽然LMD已被用于肾脏组织，但由于识别肾脏特定管段的能力减弱，这一强大工具的使用一直受到限制。在这项研究中，我们描述了使用免疫荧光LMD(IF-LMD)分离小鼠肾脏特定细胞的方法的重大改进。使用IF-LMD，我们不仅可以重复分离肾小球，还可以分离S1-S2近端节段、S3小管和粗大的升支。我们还展示了一种新的快速免疫荧光染色技术的实用性，并提供了IF-LMD的下游应用，如实时聚合酶链式反应和前沿蛋白质组学研究。这一技术突破可能成为了解异质肾脏环境中细胞和分子事件的宝贵工具。
Laser micro-dissection (LMD) is a very useful tool that allows the isolation of finite areas from tissue specimens for downstream analysis of RNA and protein. Although LMD has been adapted for use in kidney tissue, the use of this powerful tool has been limited by the diminished ability to identify specific tubular segments in the kidney. In this study, we describe a major improvement in the methodology to isolate specific cells in the mouse kidney using immunofluorescence LMD (IF-LMD). Using IF-LMD, we can reproducibly isolate not only glomeruli, but also S1-S2 proximal segments, S3 tubules, and thick ascending limbs. We also demonstrate the utility of a novel rapid immunofluorescence staining technique, and provide downstream applications for IF-LMD such as real-time PCR and cutting-edge proteomic studies. This technical breakthrough may become an invaluable tool for understanding cellular and molecular events in the heterogeneous kidney milieu.