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Signal Transduction Immunohistochemistry pp 127–141Cite as

EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1554))

Abstract

Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. The particular interest to determine intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. In this chapter, we discuss the EicosaCell protocol, an assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This chapter covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.

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Acknowledgments

The work of the authors is supported by PRONEX-MCT, Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, Brazil), PAPES-FIOCRUZ, Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ, Brazil), and NIH grants (AI022571, AI020241, AI051645).

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Authors and Affiliations

  1. Laboratório de Inflamação, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

    Christianne Bandeira-Melo, Ligia Almeida Paiva & Natália R. T. Amorim

  2. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Peter F. Weller

  3. Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil

    Ligia Almeida Paiva & Patricia T. Bozza

Authors
  1. Christianne Bandeira-Melo
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  2. Ligia Almeida Paiva
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  3. Natália R. T. Amorim
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  4. Peter F. Weller
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  5. Patricia T. Bozza
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Corresponding authors

Correspondence to Christianne Bandeira-Melo or Patricia T. Bozza .

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Editors and Affiliations

  1. Immunocytochemistry & Elispot Assays, Bio-Techne, Inc., Minneapolis, Minnesota, USA

    Alexander E. Kalyuzhny

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Bandeira-Melo, C., Paiva, L.A., Amorim, N.R.T., Weller, P.F., Bozza, P.T. (2017). EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis. In: Kalyuzhny, A. (eds) Signal Transduction Immunohistochemistry. Methods in Molecular Biology, vol 1554. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6759-9_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6759-9_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6757-5

  • Online ISBN: 978-1-4939-6759-9

  • eBook Packages: Springer Protocols

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