In resting neutrophils, these are clearly segregated, and immunofluorescence staining reveals no overlap of the signals if magnifications of 20× or higher are used given that the section thickness does not exceed 3–5 μm or confocal microscopy is used. During NETosis, NET components gradually intermingle to a homogenous mixture in late phases of NETosis and in NETs. Accordingly, fluorescence signals for nuclear and granular or cytoplasmic NET components overlap increasingly. These fluorescence signals can be segmented automatically (Figures 2C,D), and the area of signal overlap defines NETs (Figures 2B,E). In hematoxylin/eosin-stained tissue slices, NETs can appear as dark diffuse strands [Figures 2F,G and Ref. (14)], but the positive identification of these smears demands overlapping immunodetection of NET components.