Abstract
The pathogenic potential of neutrophil extracellular traps (NETs) was recently described, and their detection in tissue could serve as a prognostic marker. NETs are delicate and filigree structures; hence good tissue preservation is essential for their detection. Indeed, analysis of paraffin-embedded tissue has proven superior to the study of cryo sections. Though, under favorable conditions, the presence of NETs can be detected in tissue sections stained with histological dyes, definitive identification of NETs needs the colocalization of immunofluorescent signals for both nuclear and granular (or cytoplasmic) NET components. We tested diverse antigen retrieval methods and various combinations of commercially available antibodies and present here staining protocols to detect NETs in human and murine tissue sections.
Highlights
Neutrophil extracellular traps (NETs) probably evolved to counteract invading pathogens [1]
The cytoplasmic neutrophil extracellular traps (NETs) component calprotectin [3], a heterodimer consisting of Calgranulin A and B, is readily detected at all temperatures tested
In contrast to staining of NETs derived from isolated neutrophils stimulated in vitro, NETs in tissue are not identified, and staining for just one component, e.g., DNA, is not sufficient
Summary
Neutrophil extracellular traps (NETs) probably evolved to counteract invading pathogens [1]. Fixation with buffered paraformaldehyde solution, ideally by perfusion, preserves the tissue architecture including NETs. Here, we present methods for formalin-fixed and paraffin-embedded sections. Antigen retrieval is required that normally involves heating of the rehydrated sections in a suitable heat-induced epitope retrieval (HIER) buffer [18, 19] This breaks the methylene bridges that prevent binding of the antibody and renders the epitopes accessible. We tested a series of antibodies against NET components for their ability to bind to their epitopes in formalin-fixed paraffin-embedded tissue. We present protocols that allow simultaneous staining for nuclear and granular or cytoplasmic NET components in paraffin-embedded tissue sections after antigen retrieval.
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