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Analysis of Mouse Brain Microvascular Endothelium Using Laser Capture Microdissection Coupled with Proteomics

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Book cover The Blood-Brain and Other Neural Barriers

Part of the book series: Methods in Molecular Biology ((MIMB,volume 686))

Abstract

The blood–brain barrier (BBB) has been well studied in terms of its pharmacological properties. However, for a better understanding of the molecular mechanisms regulating these activities, means to thoroughly investigate the BBB at the genomic and proteomic levels are essential. Global gene expression analysis platforms have, in fact, provided a venue for cataloguing the BBB transcriptome. By comparison, and largely because of technical issues, there have been few comprehensive studies of the cerebral microvasculature at the protein level. Recent advances in both microdissection techniques and proteomic analytical tools have nonetheless circumvented many of these obstacles, allowing for isolation of relatively pure cell populations from complex tissues in situ and profiling of cellular proteomes. For example, immunohistochemistry-guided laser capture microdissection (immuno-LCM) provides the unique opportunity to selectively remove brain microvascular endothelial cells from the surrounding cell populations at the BBB, while supporting downstream proteomic analysis. In this chapter, we describe the use of immuno-LCM coupled with a sensitive, high resolution, hybrid linear ion trap coupled with Fourier transform mass spectrometry (FTMS) for proteomic profiling of mouse brain microvascular endothelium, a crucial cellular component of the BBB. We provide details of the quick double-immunostaining protocol for immuno-LCM, laser capture process, sample pooling, and protein recovery followed by in-gel digestion of protein sample, mass spectrometric analysis, and protein identification. Using such an approach to obtain comprehensive protein expression profiles of the cerebral endothelium in situ will enable detailed understanding of the crucial mediators of brain microvascular signaling and BBB function in both normal and pathophysiological conditions.

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Acknowledgements

This work was supported by National Institutes of Health grants RO-1-MH54718 and R21-NS057241 to J.S. Pachter. The authors thank Dr. Barry Karger of Northeastern University for application development of LCM with MS.

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Authors and Affiliations

  1. Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA

    Nivetha Murugesan, Jennifer A. Macdonald & Joel S. Pachter

  2. Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA, USA

    Qiaozhan Lu, Shiaw-Lin Wu & William S. Hancock

Authors
  1. Nivetha Murugesan
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  2. Jennifer A. Macdonald
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  3. Qiaozhan Lu
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  4. Shiaw-Lin Wu
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  5. William S. Hancock
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  6. Joel S. Pachter
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Editor information

Editors and Affiliations

  1. Banting Institute, Dept. Laboratory Med. & Pathobiology, University of Toronto, College St. 100, Toronto, M5G 1L5, Ontario, Canada

    Sukriti Nag

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Murugesan, N., Macdonald, J.A., Lu, Q., Wu, SL., Hancock, W.S., Pachter, J.S. (2011). Analysis of Mouse Brain Microvascular Endothelium Using Laser Capture Microdissection Coupled with Proteomics. In: Nag, S. (eds) The Blood-Brain and Other Neural Barriers. Methods in Molecular Biology, vol 686. Humana Press. https://doi.org/10.1007/978-1-60761-938-3_14

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  • DOI: https://doi.org/10.1007/978-1-60761-938-3_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-937-6

  • Online ISBN: 978-1-60761-938-3

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