Discussion
Due to their pathogenic potential, identification of NETs in tissue samples both from patients and from laboratory animals is important and could be of diagnostic value. In contrast to staining of NETs derived from isolated neutrophils stimulated in vitro, NETs in tissue are not easily identified, and staining for just one component, e.g., DNA, is not sufficient.
In order to allow simultaneous immunodetection of two or three NET components, we tested a series of commercially available antibodies for their property to react with NET markers in paraffin-embedded tissue. Each antibody was tested using various antigen retrieval protocols (Table 1). We use unlabeled primary antibodies from different hosts and detect the bound antibodies with species-specific secondary antibodies, which are cross-absorbed against serum proteins of a number of hosts. This avoids false-positive staining due to unspecific cross-labeling and facilitates dye swaps using different combinations of secondary antibodies. We found that good results were obtained performing antigen retrieval at neutral pH and temperatures between 37 and 50°C, which allow combinations of various antibodies against nuclear, granular, and cytoplasmic NET components (Figure 1).
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.
We found that under mild antigen retrieval conditions, antibodies against H3 (Figure 3) and H2B (Figures 4B,D) stain relaxed chromatin as present in NETs and netting neutrophils stronger than the compact chromatin of normal nuclei. This property can be used to scan at low power magnifications for areas that may contain NETs for subsequent detailed analysis using a second NET marker.
Figure 4 depicts a section of an archived sample of human brain fungal abscess stained for NE (Figure 4A, green in Figure 4D), H2B (Figure 4B, red in Figure 4D), and DNA (Figure 4C, blue in Figure 4D). Interestingly, the staining intensity for NE is very high in granules but rather low in NETs. This is probably due to differences in protein concentration. In contrast, staining for H2B is generally lower in condensed nuclei than in NETs. Presumably, the epitope of this antibody is better accessible in relaxed compared to compact chromatin. Notably, this preference for binding to decondensed chromatin depends on the temperature used for antigen retrieval: when HIER buffer is heated above 55°C, antibodies against H2B and H3 react strongly both with NETs and with compact nuclei. Apparently, antigen retrieval at higher temperatures exposes histone epitopes that are normally hidden.
Ly6G is a differentiation antigen, which is expressed in mature neutrophils. In areas of massive infiltration, neutrophils are densely packed leaving nearly no space between the cells. Under these conditions, GPI-anchored Ly6G delineating the cell membrane can come in close contact to extracellular NETs that may result in the interpretation of Ly6G-positive areas as NETs (23). It has been shown that NETs do not contain membrane proteins, and Ly6G was not found as a NET constituent (3). For proper identification of NETs in tissue, immunostaining for generally accepted NET markers as presented in this protocol should be employed.
We have identified a set of antibodies, which can be used to detect NET components in paraffin-embedded tissue both of human and murine origin. Using mild antigen retrieval protocols, many of these antibodies can be combined to yield a satisfactory signal intensity. We hope that these protocols will be useful for a more reliable detection of NETs in tissue.