Neutrophil extracellular traps (NETs) probably evolved to counteract invading pathogens (1). When their production or degradation is not controlled, they have pathogenic potential and are involved in numerous diseases including autoimmunity, thrombosis, lung diseases, infertility, diabetes, cancer, and neurodegeneration, reviewed in (2). NETs are composed of chromatin, granular, and some cytoplasmic constituents (3). In naive neutrophils, granular and nuclear antigens are spatially separated and during NETosis, some granular proteins, like neutrophil elastase (NE) and myeloperoxidase (MPO) gradually migrate to the nucleus (4, 5). Also, histones are citrullinated during NETosis (6, 7). Thus, the localization of granular proteins in the nucleus and the citrullination of histones provide unique feature for neutrophils undergoing NETosis that can be exploited for identification of these cells in tissue sections.

In vitro, there are a variety of protocols to detect and quantify NETs (8–12). It is important to note that the widely spread out strands associated with NET images generated in vitro are probably an artifact of fixation. Indeed, in life cell imaging studies with isolated neutrophils, NETs appear as a diffuse cloud formed by the strands of NETs floating in the medium (13). It is not clear how NETs appear in tissues, where space is restricted, and the NETs are unlikely to appear as the large areas observed in vitro. Classical histological stains, like hematoxylin/eosin, may indicate the presence of NETs, but there are only few examples of NETs detection with histological stains (14, 15). Notably, there is no general protocol to identify NETs in tissues.

While they are well suited for staining with most antibodies, cryo sections from freshly frozen tissue have the disadvantage that due to ice crystal formation neutrophils in the tissue can be damaged, thus NET-like structures can be generated as a preparation artifact. In contrast, fixation with buffered paraformaldehyde solution, ideally by perfusion, preserves the tissue architecture including NETs. Here, we present methods for formalin-fixed and paraffin-embedded sections. These sections are available from pathological studies and can be conserved indefinitely before analysis.

Most antibodies will not readily bind to their epitopes in formalin-fixed tissue. The reason for this is the formation of intra- and intermolecular cross-links by methylene bridges that mask most epitopes (16, 17). For successful immunohistological staining, antigen retrieval is required that normally involves heating of the rehydrated sections in a suitable heat-induced epitope retrieval (HIER) buffer (18, 19). This breaks the methylene bridges that prevent binding of the antibody and renders the epitopes accessible. In a study with histopathologically important antibodies, it was shown that most epitopes detected by clinically relevant antibodies are linear and can be reversibly blocked by binding to neighboring proteins during fixation (20).

We tested a series of antibodies against NET components for their ability to bind to their epitopes in formalin-fixed paraffin-embedded tissue. We selected nine antibodies with good staining properties and tested various antigen retrieval methods to find suitable combinations for double or triple immunofluorescence. In this paper, we present protocols that allow simultaneous staining for nuclear and granular or cytoplasmic NET components in paraffin-embedded tissue sections after antigen retrieval.

For the identification of NETs it is necessary to determine if nuclear antigens are colocalized with granular and/or cytoplasmic components. Hence, micrographs have to be prepared at a primary magnification of at least 20×. The resulting images can be used to quantify the fluorescent signals as an unbiased means for the detection and measurement of NETs in tissue.

Archived paraffin blocks of mouse lungs and of a human brain fungal abscess were used. Mouse breeding and experiments were approved by the Berlin state authority Landesamt für Gesundheit und Soziales, permit G0200/15. Pathology sample collection was approved by the ethical committee of Charité University Hospital, Berlin, Germany.

All antibodies were obtained from commercial suppliers (Table 1). The following antigen retrieval solutions were used: R-Universal Buffer pH7, 10× (Aptum APO 0530500), Target Retrieval Solution pH9 10mM Tris (TRS) 10× (Dako S236784), and Target Retrieval Solution pH6 10mM Citrate 10× (Dako S236984-3).

Fluorescence images were recorded using a Leica SP8 confocal or a Leica DMR widefield microscope (equipped with bandpass filter blocks and a Jenoptik ProgRes MF USB camera).

Complete tissue sections were digitized using a ZEISS Axioscan Z1 slide scanner.

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.

Designed study: VB, UA, CG, and AZ. Tissue immunostainings: UA. Microscopy: UA and VB. Image analysis: CG. Wrote the manuscript: VB, UA, CG, and AZ.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.