Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) were first visualized by Brinkmann et al. as a pathway of cellular death different from apoptosis and necrosis in 2004¹. In this pathway, neutrophils release their decondensed chromatin into the extracellular space to form large web-like structures covered in antimicrobial proteins that were formerly stored in the granules or cytosol. These antimicrobial proteins include neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (H3cit), which are commonly used for indirect immunofluorescence detection of NETs². This method not only identifies the quantitative presence of these proteins; indeed, it has the advantage of specifically detecting NET-like structures. In the NETs, the mentioned proteins co-localize with extracellular DNA, which can be detected by an overlap of the fluorescence signals of each stained protein and the extracellular DNA. In contrast to the overlapping signals due to extracellular DNA and protein co-localization in NETs, intact neutrophils show no co-localization. Here, the NET components are usually stored separately in the granules, nuclei, and cytosol³.

Since their first discovery, it has been shown that NETs play a central role in numerous diseases, particularly those involving inflammation. NETs show antimicrobial functions during infection through trapping and killing extracellular pathogens in blood and tissue^4,5. However, NETs have also been connected to autoimmune diseases and hyperinflammatory responses, like systemic lupus erythematosus, rheumatic arthritis, and allergic asthma^6,7,8. NETs promote vaso-occlusion and inflammation in atherosclerosis, platelet adhesion, and are speculated to play a role in metastatic cancer^9,10,11. Nevertheless, they are thought to have anti-inflammatory properties by reducing proinflammatory cytokine levels¹². While NETs are gaining more interest in a broader field of research, a robust NET detection method is fundamental for future research.

Even though the visualization of NETs in different tissue using immunofluorescence imaging is complex and requires customization, apart from electron microscopy, it is currently one of the most renowned methods for visualizing the interactions between NETs and cells and is predominantly used in formalin-fixed paraffin-embedded tissues (FFPE)^13,14. However, comparing NET imaging is difficult, as different laboratories use their own customized protocols. These protocols differ in their use of antibodies, antigen retrieval, or permeabilization method and are often optimized for a specific type of tissue^{3,13,15,16,17,18,19,20,21,22,23,24,25,26,27}.

After Brinkmann et al. published the first methodic study using immunofluorescent visualization of NETs in FFPE tissue, we wanted to optimize this protocol for a wider variety of tissues and species¹⁵. Additionally, to establish a broadly applicable immunofluorescence protocol, we tested different modified protocols from studies that used immunofluorescence methods in FFPE tissue to detect NETs^{3,13,16,17,18,19,20,21,22,23,24,25,26,27}. Furthermore, we tried a new H3cit antibody for more specific extracellular staining²⁸. We hypothesize that by systematically adapting current staining protocols to different species and tissue, in vitro imaging can be improved, resulting in a better representation of the interaction between neutrophils and NETs both locally and systemically.