Immunofluorescent Detection of NET Components in Paraffin-Embedded Tissue

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 mu m 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.