Segmentation of Areas Positive for Nuclear and Granular NET Components Allow Quantification of NETs in Tissue
Figure 2C shows a confocal image of a neutrophil-rich area of a mouse lung infected with Candida albicans and stained for NE (green, Millipore 48101) and H3 (red, ABIN 1735464) as well as for DNA (Hoechst 33342). Antigen retrieval was performed with R-Universal Buffer at 50°C. Using automatic Otsu thresholding, positive areas for both channels were depicted white, while areas below threshold were depicted black (Figures 2E,F). The overlap of both indicating the NET-positive area is shown in Figure 2G and superimposed on the tissue staining (Figure 2D).
Hematoxylin/eosin staining of the same area in a consecutive section is presented in Figures 2A,B. The overview shows infiltration of neutrophils (Figure 2B, center). At higher magnification, extracellular strands of DNA are visible (arrowheads in Figure 2B). Identification of these strands as NETs needs immunofluorescence.