Abstract
Neutrophil extracellular traps (NETs) consist of decondensed chromatin fibers studded with granular and cytoplasmic proteins and peptides that are released by stimulated neutrophil granulocytes. If present in abundance (e.g., in large thrombi), NETs are depicted in H&E-stained tissue sections as pale bluish areas. Since no NET-specific antibodies exist, to unambiguously identify even small amounts of NETs in tissue, it is essential to demonstrate colocalization of nuclear and granular/cytoplasmic NET components which in unstimulated neutrophils are clearly separated. This requires good tissue preservation and a very defined immunolocalization, which can be achieved by using 2–3 μm thick sections of paraffin-embedded tissue. It provides sufficiently good tissue preservation for subcellular localization of two or more NET components, thereby allow to differentiate stimulated from unstimulated neutrophils and to clearly identify NETs. In this chapter, we will provide protocols for antigen retrieval and immunofluorescent labeling of NET components in paraffin-embedded tissue with commercially available antibodies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Brinkmann V, Reichard U, Goosmann C et al (2004) Neutrophil extracellular traps kill bacteria. Science 303:1532–1535
Papayannopoulos V (2018) Neutrophil extracellular traps in immunity and disease. Nat Rev Immunol 18:134–147
Brinkmann V (2018) Neutrophil extracellular traps in the second decade. J Innate Immun 10:414–421
Hakkim A, Fuchs TA, Martinez NE et al (2011) Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat Chem Biol 7:75–77
Fuchs TA, Abed U, Goosmann C et al (2007) Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176:231–241
Metzler KD, Fuchs TA, Nauseef WM et al (2011) Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity. Blood 117:953–959
Papayannopoulos V, Metzler KD, Hakkim A et al (2010) Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 191:677–691
Neeli I, Khan SN, Radic M (2008) Histone deimination as a response to inflammatory stimuli in neutrophils. J Immunol 180:1895–1902
Wang Y, Li M, Stadler S et al (2009) Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol 184:205–213
Shiogama K, Onouchi T, Mizutani Y et al (2016) Visualization of neutrophil extracellular traps and fibrin meshwork in human fibrinopurulent inflammatory lesions: I. light microscopic study. Acta Histochem Cytochem 49:109–116
Robertson D, Savage K, Reis-Filho JS et al (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol 9:13
Rait VK, Xu L, O'Leary TJ et al (2004) Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment. Lab Investig 84:300–306
Yamashita S, Okada Y (2005) Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry. J Histochem Cytochem 53:13–21
Cattoretti G, Pileri S, Parravicini C et al (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:83–98
Shi SR, Imam SA, Young L et al (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43:193–201
Weckbach LT, Grabmaier U, Uhl A et al (2019) Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis. J Exp Med 216:350–368
Acknowledgments
The funding source of this work is the Max Planck Society. We thank Arturo Zychlinsky for critically reading the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Abu-Abed, U., Brinkmann, V. (2020). Immunofluorescent Detection of NET Components in Paraffin-Embedded Tissue. In: Quinn, M., DeLeo, F. (eds) Neutrophil. Methods in Molecular Biology, vol 2087. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0154-9_24
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0154-9_24
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0153-2
Online ISBN: 978-1-0716-0154-9
eBook Packages: Springer Protocols