The authors have nothing to disclose.

Tissue protocols were approved by Landesamt f&#252;r Gesundheit und Soziales, Berlin, Germany (G0121/16). Experiments were conducted in accordance with the European directive 2010/63/EU on Care, Welfare and Treatment of Animals.
1. Fixation, dehydration, paraffin embedding, sectioning, mounting of sections
<ol>
	<li>Prepare 2% formaldehyde solution by dissolving paraformaldehyde in TBS (pH 7.4). Avoid heating above 60 &#176;C. Cool to RT. Formaldehyde solution can be stored at -20 &#176;C.</li>
	<li>Place fresh tissue (here: mouse lung) into a glass Petri dish with TBS. With a scalpel, dissect into pieces not exceeding 20 mm x 30 mm x 3 mm in size. Immerse specimen into 2% formaldehyde solution in TBS. 
	NOTE: The volume of fixative should be at least 20x that of the tissue.</li>
	<li>Fix at RT for 8&#8722;20 h. Transfer specimens to TBS. Place into cassettes.</li>
	<li>Dehydrate using a series of ethanol (70%, 80%, 90%, 96%, 100%, 100%), with each step lasting 1 h.</li>
	<li>Clear specimens 2x in 100% xylene (dimethylbenzene), each step 1 h.</li>
	<li>Soak in paraffin 2x at 60 &#176;C (melting temperature 54&#8722;56 &#176;C), each step 1 h.</li>
	<li>Mount specimens using embedding molds, use the cassette bottom as a cover.</li>
	<li>Let paraffin solidify and remove embedding molds.</li>
	<li>Prepare 3 &#181;m tissue sections, and let them float on 37 &#176;C water bath.</li>
	<li>Float sections on water surface onto adhesive glass slides (Table of Materials).</li>
	<li>Incubate sections on glass slides overnight at 40 &#176;C to bond the tissue to the glass.</li>
</ol>
2. Rehydration, heat-induced epitope retrieval, staining, mounting, microscopic analysis
<ol>
	<li>Place sections on glass slides in racks and submerse them into the media used for dehydration and clearing, in reverse order, with each step for 5 min: 2x 100% xylene, ethanol series (2x 100%, 96%, 90%, 80%, 70%).</li>
	<li>Heat a water bath with a temperature-controlled hot plate to 70 &#176;C. Place a jar filled with heat-induced epitope retrieval (HIER)-buffer (pH 9; Table of Materials), containing 10% glycerol as a temperature buffer, into a water bath. When the HIER buffer has reached 70 &#176;C, place the rack with slides into buffer jar.</li>
	<li>Incubate slides for 120 min at 70 &#176;C.</li>
	<li>Remove the jar from the water bath and let it cool to RT. Rinse sections 3x with deionized water and once with TBS (pH 7.4).</li>
	<li>Carefully remove liquid from slides between sections with rolled filter paper or cotton swab, leaving sections hydrated.</li>
	<li>Create barrier around sections with a hydrophobic barrier pen.</li>
	<li>Incubate sections in blocking buffer (1% bovine serum albumin [BSA], 2% normal donkey serum, 5% cold water fish gelatin and detergents [Table of Materials] in TBS) at RT for 30 min to prevent unspecific binding.</li>
	<li>Dilute primary antibodies at a concentration of 1 &#181;g/mL in blocking buffer. 
	NOTE: For human and mouse NETs, a combination of rabbit antibody against neutrophil elastase and chicken antibody against Histone 2B can be used (Table of Materials).</li>
	<li>Place slides into a moist chamber. Remove blocking buffer and add the diluted primary antibodies without washing to the sections using sufficient volume to prevent drying. Seal moist chamber with paraffin film and incubate overnight at RT.</li>
	<li>Wash sections 3x with TBS for 5 min each.</li>
	<li>Prepare working solution of secondary antibodies in blocking buffer. For detection of primary antibodies, use secondary antibodies raised in donkey and pre-absorbed against serum proteins from multiple species (Table of Materials). Cover tissue sections with working solution of secondary antibodies. Transfer slides to moist chamber, seal with paraffin film. Incubate for 1 h at RT. 
	NOTE: Donkey anti rabbit conjugated to Cy2 and donkey anti chicken conjugated to Cy3 can be combined with a DNA counterstain.</li>
	<li>Wash sections 3x for 5 min each with TBS, then once for 5 min with deionized water.</li>
	<li>Cover sections with mounting medium (Table of Materials) and apply cover glass avoiding bubble formation.</li>
	<li>Let mounting medium solidify, then analyze immunofluorescence using a wide-field microscope with appropriate band pass filters or a confocal microscope.</li>
</ol>

<ol>
	<li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Urban, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+contain+calprotectin,+a+cytosolic+protein+complex+involved+in+host+defense+against+Candida+albicans.">Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.</a> PLOS Pathogens. 5 (10), e1000639 (2009).</li><li>Fuchs, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> Journal of Cell Biology. 176 (2), 231-241 (2007).</li><li>Papayannopoulos, V., Metzler, K. D., Hakkim, A., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+elastase+and+myeloperoxidase+regulate+the+formation+of+neutrophil+extracellular+traps.">Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.</a> Journal of Cell Biology. 191 (3), 677-691 (2010).</li><li>de Boer, O. J., Li, X., Goebel, H., van der Wal, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+smears+observed+in+H&amp;E-stained+thrombus+sections+are+neutrophil+extracellular+traps.">Nuclear smears observed in H&amp;E-stained thrombus sections are neutrophil extracellular traps.</a> Journal of Clinical Pathology. 69 (2), 181-182 (2016).</li><li>Robertson, D., Savage, K., Reis-Filho, J. S., Isacke, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+immunofluorescence+labelling+of+formalin-fixed+paraffin-embedded+(FFPE)+tissue.">Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue.</a> BioMed Central Cell Biology. 9, 13 (2008).</li><li>Rait, V. K., Xu, L., O'Leary, T. J., Mason, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+formalin+fixation+and+antigen+retrieval+with+bovine+pancreatic+RNase+A+II.+Interrelationship+of+cross-linking,+immunoreactivity,+and+heat+treatment.">Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment.</a> Laboratory Investigation. 84 (3), 300-306 (2004).</li><li>Brinkmann, V., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+NETs+in+Paraffin-Embedded+Tissue.">Immunodetection of NETs in Paraffin-Embedded Tissue.</a> Frontiers in Immunology. 7, 513 (2016).</li><li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nature Reviews Immunology. 18 (2), 134-147 (2018).</li><li>Cattoretti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+unmasking+on+formalin-fixed,+paraffin-embedded+tissue+sections.">Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections.</a> Journal of Pathology. 171 (2), 83-98 (1993).</li><li>Yamashita, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+heat-induced+antigen+retrieval:+analyses+in+vitro+employing+SDS-PAGE+and+immunohistochemistry.">Mechanisms of heat-induced antigen retrieval: analyses in vitro employing SDS-PAGE and immunohistochemistry.</a> Journal of Histochemistry and Cytochemistry. 53 (1), 13-21 (2005).</li><li>Weckbach, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midkine+drives+cardiac+inflammation+by+promoting+neutrophil+trafficking+and+NETosis+in+myocarditis.">Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.</a> Journal of Experimental Medicine. 216 (2), 350-368 (2019).</li></ol>

The funding source of this work is the Max Planck Society. We thank Arturo Zychlinsky for critically reading the manuscript and Philippe Saikali for the mouse tissue.

With growing awareness of the role of NETs during pathogenesis9, their detection in tissue from patients or experimental animals is gaining increasing importance. Paraffin-embedded tissue samples have a number of advantages compared to other tissue preparations (e.g., sections of cryopreserved specimens). The tissue preservation in paraffin-embedded samples is clearly superior, and once embedded, the samples are preserved for decades, enabling retrograde studies. For detection of NETs, which are filigree structures, good sample conservation is a precondition ruling out the use of cryopreserved material, which is prone to tissue damage due to ice crystal formation that can give rise to artifacts morphologically resembling NETs.
For optimal preservation, tissue from experimental animals should be fixed shortly after death, ideally by perfusion, to avoid autolysis. As fixative, freshly prepared or freshly thawed solutions of paraformaldehyde in a suitable buffer like TBS or PBS ore optimal. This is chemically defined in contrast to formalin preparations used for standard histology, and induces less tissue autofluorescence. In contrast, human tissue often is not fixed directly after excision, and as fixative, normally a 10% dilution of formalin is utilized which contains 10% to 15% of methanol as a stabilizer to prevent polymerization, as well as formic acid, other aldehydes, and ketones. Often, the tissue is stored in this fixative for extended amounts of time before embedding. The result can be autolysis (depending on the time between excision and fixation), and excessive formation of methylene bridges due to the overfixation. Formaldehyde fixation induces changes in the tertiary structure of proteins by formation of intra- and inter-molecular methylene bridges10. Non-proteinaceous cell components like nucleic acids, carbohydrates and lipids are not fixed directly, but immobilized in the three-dimensional protein network. For successful labelling, methylene bonds have to be broken to expose the epitopes in the process of antigen retrieval. This is accomplished by heating of tissue sections mounted onto microscope slides in a suitable heat-induced antigen retrieval buffer (HIER buffer)11. Our previous study analyzed the influence of pH and temperature of HIER buffers on the detection of NET components in paraffinized tissue, and it was found that successful tissue pretreatment for one component often is suboptimal for a second component8.
In the meantime, it has been found that heating the tissue to 70 &#176;C in HIER buffer at a pH of 9 is a good compromise for many NET components and will retain good tissue preservation, which is often compromised at higher temperatures. It is proposed to use the retrieval time indicated as a starting point, but especially with archival specimens of unknown fixation parameters, the staining intensity can be unsatisfactory. In this case, prolongation of or higher temperature during the retrieval procedure can improve staining efficiency. This can also influence the histone 2B staining of the IgY antibody used in our protocol. As shown in Figure 1, decondensed chromatin can be found in neutrophils undergoing NETosis as well as in NETs stains much stronger with this antibody compared to chromatin in resting neutrophils. This is probably due to restricted access of the 180 kDa IgY molecule to compact nuclei and may differ after increased epitope recovery. This may intensify binding efficiency even in areas of condensed chromatin, which will result in stronger fluorescence in normal nuclei of neutrophils and other cells. The difference in staining efficiency between neutrophils undergoing NETosis and non-stimulated neutrophils could be less pronounced than depicted in Figure 1. Areas of NET formation still would be identified by the co-localization of NE, H2B, and DNA.
The fixation procedure can also induce significant autofluorescence of the tissue, mainly in the bluish/greenish part of the spectrum. It is important to avoid most of this autofluorescence by using adequate narrow bandpass fluorescence filter sets (wide-field) or detector settings (confocal) that match the emission maximum of the fluorochrome used with blue excitation, e.g., Cy2, Alexafluor 488. In extreme cases of autofluorescence, the bluish/greenish part of the spectrum should be avoided. Instead, fluorescence signals can be detected easier in the far red part of the spectrum (e.g., secondary antibodies coupled to Cy 5 or Alexafluor 635), but this requires filterless black/white cameras or confocal microscopes. Since the human eye is rather insensitive beyond 600 nm, far red fluorescence signals can hardly be detected using oculars. In any case, it is important to use negative controls (e.g., non-immune sera/isotype controls instead of primary antibodies or omitting primary antibodies) to determine suitable exposure times or detector settings.
Tissue sections of 1&#8722;2 &#181;m can be analyzed with wide-field microscopes using 10x or 20x objectives. These lenses provide a large focal depth, so (nearly) the entire tissue section will be in focus. This can be used to quickly scan tissue sections for areas of NET formation if they are sufficiently large (as in Figure 1). The colocalization of the green (NE), red (H2B), and blue (DNA) channels results in a white staining indicating areas of NET formation (Figure 1G). For higher magnifications, confocal or wide-field microscopes with deconvolution are necessary. Figure 2 shows details of the human appendicitis specimen also used for Figure 1. In Figure 2A, NE localizes to small dots (granules), but a large proportion is extracellular, often forming stripes which overlap with H2B (Figure 2B) and DNA (Figure 2C). This colocalization results in a whitish overlay indicating NETs (Figure 2D). A very similar pattern can be found in the lung section of a mouse infected with M. tuberculosis (Figure 3).
The specimens presented here are characterized by areas with a high degree of NET formation which can be identified even at low magnification. Depending on the tissue density and respective stimulus, NET formation can be much less pronounced, down to NET formation of small groups of neutrophils (for an example in myocarditis, see a previous publication12). It is believed that this protocol will help foster more research on NETs in tissue and hopefully aid in unravelling unrecognized roles of NETs in the formation or prevention of diseases.

Neutrophil extracellular traps (NETs) have a complex three-dimensional structure. High resolution scanning-electron microscopic analysis revealed that they consist of smooth fibers with a diameter of 15&#8722;20 nm (chromatin) studded with globular domains of &#62;25 nm which presumably consist of an assortment of about 30 granular and cytoplasmic proteins and peptides1,2. When generated in vitro from isolated neutrophils, NETs can be identified by staining of two or more of their components by immunofluorescence (e.g., histones and neutrophil elastase [NE]). In unstimulated polymorphonuclear leukocytes (PMN), histones are located exclusively in the nucleus, while NE is contained in granules. During NETosis, NE enters the nucleus, where it processes histones3,4. NETs are characterized by colocalization of components, which are clearly separated in unstimulated neutrophils. Since currently no antibodies exist that exclusively detect NET specific epitopes (i.e., do not react with na&#239;ve neutrophils), detecting colocalization of neutrophil proteins in NETs is the only way to identify NETs.
In tissue, NETs can hardly be detected by conventional histological stains (e.g., by staining with hematoxylin/eosin [H&#38;E], which depicts NETs as a diffuse pale bluish tinge). Only when highly abundant, NETs can clearly be distinguished with H&#38;E staining, such as in thrombi5. Since NETs are diffuse per se, tissue structure has to be preserved optimally, so cryo-preparations that are prone to induce freezing artifacts are suboptimal for analysis of NETs in tissue. Instead, formaldehyde fixation and paraffin embedding has been shown to preserve NET structure in tissue well2. (Immuno-)histological inspection of sections of paraffin-embedded tissue is the standard method for pathological analysis. As tissue in paraffin blocks is conserved even at room temperature (RT), tissue specimens from daily diagnostic work can be compared with specimens that have been prepared years ago, with questions and techniques that have recently arisen. NETs in tissue have not been detected until recently, and detailed analysis of NET formation and removal during the course of diseases may lead to new insights into pathogenesis. The use of paraffin-embedded tissue has the invaluable advantage to allow the analysis of archival material and to conduct retrospective studies6. Undeniably, these benefits come at a cost. Before embedding into paraffin, tissue has to be formaldehyde-fixed, dehydrated and heated above 50 &#176;C. These procedures induce tissue autofluorescence as well as epitope masking.
To limit fixation artifacts, fixation time should be kept to a minimum, so the size of tissue samples should not exceed an area of 20 mm x 30 mm with ~3 mm thickness. Samples of this size are completely fixed after overnight incubation at RT in formaldehyde, ideally followed by direct dehydration and paraffin embedding. Alternatively, fixed samples can be stored for 1 or 2 days in buffer. For immunofluorescence studies, the fixative should be freshly prepared from paraformaldehyde dissolved in a suitable buffer (e.g., phosphate-buffered saline [PBS] or Tris-buffered saline [TBS]). During fixative preparation, the temperature must be kept below 60 &#176;C to prevent formation of formic acid. Formalin, which is a standard fixative for pathology, should not be used since it contains methanol, other aldehydes, ketones, and formic acid. These impurities lead to increased epitope masking and significant tissue autofluorescence.
For successful immunolabelling, epitope masking has to be reverted in a process called antigen retrieval. Tissue sections are heated in a suitable buffer, which is believed to break methylene bridges, so epitopes become accessible to the antibodies7. For detection of NETs, heat-induced epitope retrieval (HIER) is preferred to other methods such as the use of proteolytic enzymes. Routinely, immunolabelling of paraffin sections is carried out with a single antibody followed by a peroxidase-labelled secondary antibody, which is detected with a precipitating substrate. Compared to immunofluorescence, enzyme-based detection methods have a lower spatial resolution due to diffusion of the substrate precipitate, and generally, simultaneous detection of more than one antigen is limited6.
As currently no antibodies exist that exclusively bind to NETs, this protocol is used to label two proteins, histone 2B, which is nuclear, and neutrophil elastase, which is localized in granules. In unstimulated neutrophils, these proteins are separated, but they are colocalized in neutrophils undergoing NETosis and in NETs. The simultaneous detection of two antigens can be achieved with two primary antibodies raised in different hosts and two species-specific secondary antibodies labelled with different fluorochromes. This report is a standardization of our previously published protocol8 and uses a combination of two commercially available antibodies that reliably stain NET components in paraffin-embedded tissue, both fresh and archival, of human and murine origin.

<table><tbody><tr><td>bovine serum albumine</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>chicken anti Histone 2B antibody</td><td>Abcam</td><td>ab 134211</td><td></td></tr><tr><td>cold water fish gelatine</td><td>Sigma-Aldrich</td><td>G7765</td><td></td></tr><tr><td>donkey anti chicken antibody, Cy3 conjugated, for multiple labelling</td><td>Jackson Immuno Research</td><td>703-165-155</td><td></td></tr><tr><td>donkey anti rabbit antibody, Cy2 conjugated, for multiple labelling</td><td>Jackson Immuno Research</td><td>711-225-152</td><td>alternatively Cy5 conjugate, 711-175-152</td></tr><tr><td>embedding cassettes</td><td>Roth</td><td>K116.1</td><td></td></tr><tr><td>embedding molds</td><td>Sakura</td><td>4162</td><td></td></tr><tr><td>embedding station</td><td>Microm</td><td>AP280</td><td></td></tr><tr><td>ethanol</td><td>Roth</td><td>9065.4</td><td></td></tr><tr><td>filter paper, rolled</td><td>OCB</td><td></td><td></td></tr><tr><td>forceps</td><td>Dumont</td><td>7a</td><td></td></tr><tr><td>glass cylinder with 20 cm diameter</td><td>VWR</td><td>216-0075</td><td></td></tr><tr><td>glycerol</td><td>Roth</td><td>3783.1</td><td></td></tr><tr><td>HIER buffer pH 9</td><td>Scytek</td><td>TES999</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>14533</td><td></td></tr><tr><td>incubator (dry, up to 40 &amp;deg;C)</td><td>Memmert</td><td>MMIPP30</td><td></td></tr><tr><td>moist chamber (plastic box with tight lid)</td><td>Emsa</td><td>508542</td><td></td></tr><tr><td>Mowiol 40-88</td><td>Aldrich</td><td>32.459-0</td><td></td></tr><tr><td>normal donkey serum</td><td>Merck</td><td>S30</td><td></td></tr><tr><td>PAP-pen ImmEdge</td><td>Vector Lab</td><td>H-4000</td><td></td></tr><tr><td>paraffin microtome</td><td>Microm</td><td>355S</td><td>water bath included</td></tr><tr><td>paraformaldehyde</td><td>Aldrich</td><td>16005</td><td></td></tr><tr><td>petri dishes</td><td>Schubert /Weiss</td><td>7020051</td><td></td></tr><tr><td>rabbit anti ELANE antibody</td><td>Atlas</td><td>HPA068836</td><td></td></tr><tr><td>racks, jars for microscope slides</td><td>Roth</td><td>H554.1 H552.1</td><td></td></tr><tr><td>scalpel</td><td>Braun</td><td>Fig.22</td><td></td></tr><tr><td>Superfrost Plus glass slides</td><td>Thermo Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>temperature-controlled hot plate</td><td>NeoLab</td><td>D6010</td><td></td></tr><tr><td>tissue processor for paraffin embedding</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Tris buffered saline TBS</td><td>VWR</td><td>788</td><td></td></tr><tr><td>Triton X100</td><td>Roth</td><td>3051.4</td><td></td></tr><tr><td>Tween 20</td><td>Fluka</td><td>93773</td><td></td></tr><tr><td>water bath 37 &amp;deg;C</td><td>GFL</td><td>1052</td><td></td></tr><tr><td>widefield or confocal microscope</td><td>Leica</td><td>DMR, SP8</td><td></td></tr><tr><td>xylene (dimethylbenzene)</td><td>Roth</td><td>9713.3</td><td></td></tr></tbody></table>

immunofluorescence labelling of human and murine neutrophil extracellular traps in paraffin-embedded tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Using this protocol, NET components can successfully be detected in paraffin-embedded tissue both of human and murine origin. In unstimulated neutrophils, H2B is located exclusively in the nucleus and neutrophil elastase in granules; consequently, their fluorescence signals do not overlap. In contrast, during NETosis and after NET formation, NE, H2B, and DNA partly colocalize. If imaged as green, red, and blue signals, areas of colocalization are depicted as whitish overlay (Figure 1G, Figure 2D, and Figure 3D). This overlay can also be quantified using image analysis software. Pixels from overlapping signals that are positive for green, red, and blue have been used to create a purple overlay depicting NET areas in Figure 1G&#8217;, Figure 2D&#8217;, and Figure 3D&#8217;. Some of the cells in Figure 1 and Figure 2 have lobulated nuclei but are negative for NE. It is believed that these cells are eosinophil granulocytes.
If the tissue sections have a thickness of 2&#8211;3 &#181;m, they can be analyzed by wide-field microscopy using 10x or 20x objectives. An example is presented in Figure 1, which depicts a section of human appendicitis tissue stained for NE (green), H2B (red) and Hoechst 33342 (blue). Panels A, C, E, and G are from an area of the section containing NETs, while panels B, D, F, and H are from a different area of the same section, which contains numerous neutrophils (Figure 1B, NE) but no NETs. Areas with massive NET formation can easily be found even at low magnifications, since all three NET components colocalize, often in stringy extracellular structures, which in the overlay of the three channels appear as whitish extracellular fibers (Figure 1G), purple overlay in Figure 1G&#8217;.
The staining patterns of both tissue areas are clearly different, with NE contained in granules (Figure 1B) and DNA in nuclei (Figure 1F). Interestingly, the staining for H2B is rather weak in neutrophil-rich areas (Figure 1D) compared to NET-containing tissue (Figure 1C). This could be due to the size of the antibody (IgY has 180 kD compared to 150 kD for IgG), which may prevent binding to compact chromatin in intact nuclei, while access to H2B is facilitated if the chromatin is decondensed as is the case in NETs (Figure 1C).
For higher resolution, confocal microscopes or widefield microscopes with deconvolution have to be used to minimize out-of-focus blur. Figure 2 depicts a NET-rich area from the same human appendicitis specimen. It is a maximum projection of a confocal stack. NE (green, Figure 2A) is found in granules but is also abundant extracellularly, where it colocalizes with H2B (red, Figure 2B) and DNA (blue, Figure 2C). The extracellular colocalization results in a whitish color combination (Figure 2D). These pixels positive for green, red, and blue have been used to create a purple overlay presenting NETs in Figure 2D&#8217;.
Figure 3 is a detail of a central section from a mouse lung infected with Mycobacterium tuberculosis (M. tb). Antigen retrieval and staining conditions are the same as for Figure 1 and Figure 2. Again, colocalization of all three NET components is clearly visible as whitish areas between neutrophils, which has been used to create a purple layer indicating NETs in Figure 3D&#8217;.
The specificity of the staining is demonstrated in Supplementary Figure 1, which depicts negligible staining with control antibodies, and Supplementary Figure 1A,B depicts rabbit non-immune serum (green) and chicken anti-GFP IgY (red). Figure 1C,D shows the staining with secondary antibodies alone, the combination was the same as was used in Figure 1, Figure 2, and Figure 3. Supplementary Figure 1A,C shows a section of human appendicitis similar to Figure 1 and Figure 2. Supplementary Figure 1B,D is mouse lung tissue similar to Figure 3. DNA is stained with Hoechst 33342. Scale bar represents 25 &#181;m.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60115/60115fig01.jpg" /> 
Figure 1: Wide-field fluorescence microscopy of a paraffin section of a human appendicitis sample. 
Panels A, C, E, and G depict a tissue area with NETs, while panels B, D, F, and H show a different area of the same section that is rich in neutrophils but without NET formation. Staining is against NE (A,B; green), H2B (C,D; red), and DNA (E,F; blue). Figure 1G,H represents the overlay of all three channels. Pixels with an intensity between 80 and 256 in all colors represent areas of overlapping staining for NE, H2B, and DNA and are considered to be derived from NETs or neutrophils undergoing NETosis. These pixels have been pseudo-colored as purple in panel G&#8217;, where they form a large area; and in panel H&#8217;, where only small spots are found. Images were taken with a wide-field microscope using a 20x objective, and scale bar represents 25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60115/60115fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60115/60115fig02.jpg" /> 
Figure 2: Confocal fluorescence microscopy of NET components in a human appendicitis sample. 
The same tissue section used in Figure 1 was used. (A) Staining against NE, (B) Depiction of H2B, and (C) Hoechst 33342 staining of DNA. (D) The overlay of all three channels. Colocalization of all three signals is pseudo-colored purple in panel D&#8217;. For this, pixels with an intensity &#62;80 in all three colors were detected using Volocity 6.3. The purple area depicts NETs or neutrophils undergoing NETosis. The images were taken with a confocal microscopy as Z-stacks and presented as maximum projection. Scale bar represents 25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60115/60115fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60115/60115fig03.jpg" /> 
Figure 3: Confocal fluorescence microscopy of NET components in a mouse lung infected with M. tb. 
It is a detail from the central part of a section of a complete lung with massive neutrophil infiltration. (A) NE staining, (B) H2B staining, and (C) DNA staining. (D) The overlay of all three channels. The purple overlay in panel D&#8217; indicates pixels with intensity values of &#62;80 in all three colors indicating neutrophils undergoing NETosis as well as NETs. The images were taken as a Z-stacks with a confocal microscope and presented as maximum projection. Scale bar represents 25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60115/60115fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Staining control with unrelated primary antibodies. Human (A,C) and murine tissue (B,D) as was used for Figure 1 and Figure 2, and Figure 3, respectively, were stained with unrelated primary antibodies (A,B) or without primary antibodies (C,D). As control primary antibodies in panels A and B, serum from a non-immunized rabbit and a chicken IgY against GFP were applied. Secondary antibodies were the same as shown in all other staining and also used in panels C and D. As expected, in all conditions, negligible background staining was detected, illustrating the specificity of the antibodies used for Figure 1, Figure 2, and Figure 3. The images were taken with a confocal microscope, DNA stained with Hoechst 33342, and scale bar represents 25 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60115/60115supfig01large.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue at JoVE.com

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

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Nanyang Technological University

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JoVE Journal

Medicine

13.1K Views.  Max Planck Institute for Infection Biology. Vandaag willen we jullie varianten laten zien van het gemeenschappelijke warmte-geïnduceerde antigeenre retrieval protocol. Het combineert de voordelen van lagere temperatuur voor het neutrofiele elastase-antigeen en een hoge pH-waarde die nodig is voor histonen. Deze techniek maakt het mogelijk om NETs, neutrofiele extracellulaire vallen, in paraffineweefsels zowel van muis als mannen te bestuderen.En het wordt ook beschouwd als gearchiveerde materiaal of retrospectieve studie te bes...

Video: Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.

Telomere and telomerase play essential roles in ageing and tumorigenesis. The goal of this protocol is to show how to generate two murine inducible telomerase knock-in alleles and how to utilize them in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Immunology and Infection

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Author Spotlight: Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...

Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils

Source: Arends, E. J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59150">A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates the induction and visualization of neutrophil extracellular traps or NETs in pre-labeled neutrophils using patient serum containing anti-neutrophil cytoplasmic antibodies. Furthermore, using an impermeable DNA dye and fluorescence microscopy enables the visualization of NETs.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

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