Suitable Combinations of Antibodies and Antigen Retrieval Methods
Table 1 summarizes the results of immunofluorescence staining for NET components and neutrophil marker proteins under different antigen retrieval conditions. Figure 1 shows immunofluorescence images resulting from different antigen retrieval protocols. Immunodetection of NE is dependent on the incubation temperature, which may not exceed 60°C (Figure 1C). Staining is stronger at pH6 compared to pH9. The cytoplasmic NET component calprotectin (3), a heterodimer consisting of Calgranulin A and B, is readily detected at all temperatures tested. As expected, the staining patterns for both subunits did not differ.
Figure 1  Representative images of antibody stainings in sections of a paraffin-embedded Candida albicans-infected mouse lung. Different antigen retrieval methods were used (details in Table 1). Antibodies were against NE (A–C), H3 (D–F), citrullinated H3 (G–I), and H2B (J–L). Bar represents 100 μm. We tested various antibodies against histones. Only the ones that gave reliable immunostaining with NETs are specified in Table 1. Interestingly, both antibodies against H3 and H2B produced different staining patterns depending on the antigen retrieval temperature. Incubation of the sections at temperatures above 55°C resulted in a strong staining of NETs and nuclei (Figures 1F,I). In contrast, at temperatures between 37 and 50°C, both antibodies reacted predominantly with relaxed chromatin in netting neutrophils and NETs, while normal neutrophils and other cells show a weak nuclear staining (Figures 1D,E,J,K; also Figure 3A). A similar staining pattern has been described for an antibody against a subnucleosomal complex in NETs derived in vitro from isolated neutrophils (13). Taken together, this difference in staining is probably due to the compaction of chromatin and the state of the antigen detected. Importantly, antibodies against citrullinated H3 (H3cit) reacted at all temperatures tested, and the staining pattern was nearly exclusively in areas with netting neutrophils and NETs (Figures 1G–I) (6, 7).
To clearly identify NETs in tissue, colocalization of granular and nuclear components has to be detected. We chose antibodies against NE and either H3 or H2B in combination with detection of citrullinated H3. As a compromise for the different conditions of antigen retrieval, we chose Buffer R-Universal at neutral pH, which allows simultaneous immunodetection of histones and NE. At magnifications of 20× or higher, the resulting images can be used for automatic detection of NET-containing areas in tissue (Figure 2C).
Figure 2  NETs in a Candida-infected mouse lung; (A,B) hematoxylin/eosin staining of a Candida albicans-infected mouse lung, consecutive section to the one used in (C–G). Areas of extracellular DNA are present (arrowheads), which stain for NET markers (C). (C) Merged fluorescence images showing DNA: blue, NE immunolabel: green, H3 immunolabel: red; (E,F) binarized images after Otsu thresholding, (E) NE above threshold, (F) H3 above threshold, (G) NETs [intersection of both, NE ∧ H3; outline of this area is superimposed in (D)]; (D) magnified section of the merged fluorescence image with outline of NETs segmentation (excluding areas <30 pixels). Bars represent 100 μm.