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Physiol Rep. 2015 Feb 12;3(2). pii: e12306. doi: 10.14814/phy2.12306. Print 2015 Feb 1.

Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis.

Author information

1
Division of Nephrology, Indiana University and the Roudebush Indianapolis VA Medical Center, Indianapolis, Indiana.

Abstract

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.

KEYWORDS:

2D‐DIGE; laser micro‐dissection; mass spectrometry; proteomics

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