This research was founded by the German Research Society (BO5534). We thank Antonia Kiwitt, Moritz Lenz, Johanna Hagens, Dr. Annika Heuer, and PD Dr. Ingo K&#246;nigs for providing us with samples. Additionally, the authors thank the team of the UKE Microscopy Imaging Facility (Core facility, UKE Medical School) for support with the immunofluorescence microscopy.

This study included mouse tissues derived from experiments approved by the Hamburg State Administration for Animal Research, Beh&#246;rde f&#252;r Justiz und Verbraucherschutz, Hamburg, Germany (73/17, 100/17, 94/16, 109/2018, 63/16). The tissues used were mouse lung and colon from a septic model and burned skin. We used 8 week old male and female mice. The European Directive 2010/63/EU on the protection of animals used for scientific purposes was followed for all the experiments. The anonymized human samples included tissues from neonatal enterocolitis, burned skin, biliary atresia, spondylodiscitis, and myocardium. According to the Medical Research Ethics Committee of Hamburg, the samples did not need informed consent, but the study was approved by the committee (WF-026/21).
1. Sample fixation
<ol>
	<li>Use the following protocol derived from Abu Abed and Brinkmann for sample fixation, dehydration, paraffin embedding, sectioning, and mounting3,15.
	<ol>
		<li>Prepare 4% formaldehyde solution by dissolving 40 g of paraformaldehyde (PFA) in 800 mL of Tris-buffered saline, pH 7.4 (TBS).</li>
		<li>Stir the mixture at 60 &#176;C under a fume hood until the PFA has dissolved. Bring the solution to room temperature (RT), and adjust the volume with TBS to 1,000 mL.</li>
		<li>Adjust the pH to 7.4. Store at 4 &#176;C for 2-3 weeks or at &#8722;20 &#176;C for up to 1 year.</li>
	</ol>
	</li>
	<li>For sample fixation, place fresh tissue in TBS buffer, and dissect into pieces smaller than 20 mm x 30 mm x 3 mm. Immerse the tissue sections in 4% paraformaldehyde solution for 12-24 h. 
	&#8203;NOTE: The original protocol used a 2% PFA solution and a fixation time of 8-20 h. With this method, some tissue sections were not completely fixated after 20 h, so the PFA concentration was increased to 4% PFA.</li>
	<li>Transfer the samples into labeled tissue processing cassettes. Use solvent-resistant markers or pencils for labeling.</li>
	<li>Start the sample dehydration by immersing then cassettes in 70% ethanol for 1 h. Then, immerse in 80% ethanol, 90% ethanol, 96% ethanol, and twice in 100% ethanol, with each step lasting 1 h.</li>
	<li>Immerse twice in 100% xylene (dimethylbenzene) and then twice in 60 &#176;C paraffin for 1 h each time.</li>
	<li>Use embedding molds for mounting, and let the paraffin solidify before removing the mould.</li>
	<li>Use a microtome for cutting 3 &#181;m thick tissue sections.</li>
	<li>Use tweezers to lay the cut sections in a 37 &#176;C water bath, and let them float on the surface to stretch the tissue cuts.</li>
	<li>Place the sections onto adhesive glass slides (Table of Materials), and let them dry overnight in a 40 &#176;C heat chamber.</li>
</ol>
2. Sample rehydration
<ol>
	<li>For deparaffinization, stack the slides in a slide rack, and submerge twice for 5 min each in the xylene replacement medium limonene (Table of Materials) and once for 5 min in a 1:1 limonene/ethanol mixture. 
	CAUTION: Limonene is flammable at 50 &#176;C and can cause allergic reactions. Use under a fume hood with eye protection and gloves. Keep away from open fire.</li>
	<li>Rehydrate the samples in a descending ethanol series by submerging the slide rack twice in 100% ethanol and once in 96% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 min each.</li>
	<li>Submerge the slide rack for 5 min in deionized water (DI water) to clean off any remaining ethanol.</li>
</ol>
3. Autofluorescence blocking and antigen retrieval
<ol>
	<li>Prepare pH 6 citrate target retrieval solution (TRS) (Table of Materials) according to the datasheet. This TRS is concentrated 10 times. Therefore, dilute 1:10 with DI water. Preheat in a plastic staining jar to 96 &#176;C. 
	NOTE: TRS breaks the methylene bridges formed during sample fixation to expose the antigen epitopes. This allows the primary antibodies to bind their target antigen. Store concentrated TRS at 2-8 &#176;C for 8 months. Always prepare fresh TRS.</li>
	<li>Take the slides out of the slide rack, and place them in a wet chamber. Pipette one to two drops of autofluorescence-reducing agent (Table of Materials) onto each sample. Incubate for 5 min. 
	NOTE: The autofluorescence-reducing agent blocks the inherent tissue autofluorescence caused by endogenous fluorophores and fixatives. Store the autofluorescence-reducing agent at RT for up to 1 year. 
	NOTE: Work only with small sections of tissues to avoid drying out of the samples or exceeding incubation time.</li>
	<li>Stack the slides back into the slide rack, and rinse for 1 min in 60% ethanol using an upward and downward motion until all the unused autofluorescence-reducing reagent has been washed off.</li>
	<li>Submerge the slide rack for 5 min in DI water.</li>
	<li>For antigen retrieval, transfer the slides into a staining jar with the preheated TRS. Incubate for 10 min at 96 &#176;C in a water bath. 
	NOTE: The 96 &#176;C water bath can be substituted with a microwave or steam cooker if the TRS is preheated to 96 &#176;C and 10 min of incubation time at 96 &#176;C is ensured.</li>
	<li>Take the staining jar out, and let it cool slowly to RT for approximately 60-90 min. 
	NOTE: The protocol can be paused here for up to 4 h, leaving the slides in the TRS.</li>
	<li>Remove the TRS, but keep the slides in the jar. Rinse twice with Tris-buffered saline with 0.05% Tween (TBST, pH 7.4) for 3 min each to wash off any leftover TRS residues.</li>
	<li>For permeabilization, prepare 0.2% Triton in phosphate-buffered saline (PBS), pH 7.4, and fill it into the staining jar to permeabilize the samples for 10 min. 
	NOTE: Permeabilization enhances the binding of the primary antibodies with the intracellular target proteins in the fixed samples.</li>
	<li>Rinse the slides again twice in TBST for 3 min each time.</li>
	<li>Prepare the wet chamber, and lay down slides. Wipe excess water from slides with paper tissues. Circle every sample with a hydrophobic barrier pen to keep the antibody solution from running off. 
	NOTE: Do not let the samples dry out. It is best to work with small sections.</li>
</ol>
4. Blocking nonspecific antibody binding
<ol>
	<li>Take ready-to-use blocking solution with donkey serum (Table of Materials), and pipette one drop onto every sample. Incubate for 30 min at RT. 
	NOTE: This blocking step minimizes the binding of the secondary antibody to nonspecific binding sites on the sample. After blocking these nonspecific epitopes and applying the primary antibody, the secondary antibody will bind to the primary antibody and not interact with the surface of the tissue. This ready-to-use blocking reagent is stored at 2-8 &#176;C for 6 months.</li>
	<li>Remove excess blocking solution from slides by tapping the edge of the slides against a hard surface. Do not rinse them.</li>
</ol>
5. Primary antibody
<ol>
	<li>Dilute primary antibodies in antibody dilution buffer (Table of Materials). Use approximately 100 &#181;L of the final solution for every sample. For the NE and H3cit (R8) antibody&#160;and isocontrol, dilute to a concentration of 5 &#181;g/mL. For MPO&#160;and&#160;corresponding isocontrol, use 10 &#181;g/mL. Prepare one tube for every primary antibody and one for every isocontrol antibody. 
	NOTE: H3cit (R8)/MPO or NE/MPO and their isocontrols can be combined for NETs staining. For the combination of H3cit (R8)/MPO, dilute to a final concentration of 5 &#181;g/mL for H3cit (R8) and 10 &#181;g/mL for MPO to&#160;a final volume of 100 &#181;L per sample. For NE/MPO, dilute to a final concentration of 5 &#181;g/mL for NE and 10 &#181;g/mL for MPO to a final volume of 100&#160;&#181;L per sample. For&#160;isocontrol, use the same concentration as for each corresponding antibody. Always use a new pipette tip for each antibody used. Primary&#160;antibodies can be stored at 4 &#176;C for 1-2 weeks and at &#8722;20 &#176;C or &#8722;80 &#176;C for up to 1 year.</li>
	<li>With two samples on one slide, use one for the isocontrol antibodies and one for the MPO/H3cit or MPO/NE antibody dilution. Use approximately 100 &#181;L for every sample, and spread the antibody solution evenly. 
	NOTE: Do not let samples dry out. Be careful to avoid mixing up the isocontrol and primary antibodies.</li>
	<li>Store in a wet chamber overnight at 4 &#176;C.</li>
	<li>The next day, tap off the excess primary antibody solution from the slides, and stack the slides in a cuvette. Rinse three times for 5 min each time with TBST to wash off the remaining primary antibody solution.</li>
</ol>
6. Secondary antibody
<ol>
	<li>Prepare the secondary antibody solution. Use two different fluorescent secondary antibodies: donkey-anti-goat for MPO and donkey-anti-rabbit for H3cit staining. Dilute each one to a concentration of 7.5 &#181;g/mL with antibody dilution buffer (Table of Materials). 
	NOTE: For double-staining, use two fluorescent antibodies with different excitation areas and minimal spectral overlap. Combine both secondary antibodies, and dilute to a final concentration of 7.5 &#181;g/mL for each antibody for a final volume of 100 &#181;L per sample. Protect the antibodies from light. The secondary antibodies can be stored at 2-8 &#176;C for 6-8 weeks or at &#8722;80 &#176;C for up to 1 year.</li>
	<li>Keep the slides in the wet chamber, and add 100 &#181;L of the secondary antibody solution onto every sample. Let them incubate for 30 min at RT and protected from light.</li>
	<li>Tap off the excess secondary antibody solution, and stack the slides in the slide rack. Submerge three times for 5 min each in a staining jar filled with PBS to wash off any remaining unbound antibodies.</li>
	<li>Prepare DAPI (4&#39;,6-diamidino-2-phenylindol) solution (Table of Materials), and dilute to a concentration of 1 &#181;g/mL with DI water. Submerge the slide rack in a staining jar with DAPI solution, and incubate for 5 min at RT in the dark. 
	NOTE: DAPI is used as a fluorescent stain for double-stranded DNA. The prepared DAPI solution can be used multiple times. Store the prepared DAPI solution at RT. The DAPI concentrate can be stored at &#8722;20 &#176;C for up to 1 year.</li>
	<li>Submerge the slide rack for 5 min in a staining jar with PBS to wash off the excess DAPI solution.</li>
</ol>
7. Mounting and storing the samples, microscopic analysis
<ol>
	<li>Mount the samples with coverslips and mounting medium (Table of Materials). 
	NOTE: Use only small amounts of mounting medium, and wipe off the excess medium with wet tissues. Wear gloves, and avoid accidentally wiping off any medium from the coverslips or slides. Avoid bubble formation, and use cotton swabs to press gently on the coverslips to remove any bubbles from the slides.</li>
	<li>For imaging, use a widefield microscope or a confocal microscope. 
	NOTE: Take an image of the isocontrol first. Lower the exposure time for the fluorescence filters (except the blue filter for DAPI) until almost no signal can be detected. Then, use this setting to examine the corresponding samples. Look for areas where a fluorescent signal can be detected in every individual sample using the fluorescence channel. After taking an image of the area, the program generates a compound image of all the channels and shows the different fluorescence signals as an overlap of different colors. The image can then be further analyzed for co-localization with imaging software such as ImageJ.</li>
	<li>Store the slides at 4 &#176;C for up to 6 months.</li>
</ol>

Staining and Imaging to Visualize Neutrophil Extracellular Traps

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<li>Risso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte+antimicrobial+peptides%3A+Multifunctional+effector+molecules+of+innate+immunity%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">Leukocyte antimicrobial peptides: Multifunctional effector molecules of innate immunity.</a> Journal of Leukocyte Biology. 68 (6), 785-792 (2000).</li>
</ol>

The authors have nothing to disclose.

In this work, we aimed to adapt and optimize the existing protocols for imaging NETs to more tissue types, beginning with the actual staining process. The first critical step for this method is the selection of the most suitable antibodies. For NE, we tried an NE antibody from a mouse host on human tissue, which showed no reliable staining compared to NE from a rabbit host. Furthermore, Th&#229;lin et al. proposed H3cit (R8) as a more specific antibody for extracellular staining. We compared this antibody with the widely used triH3cit (R2, R8, R17). Our study showed that the H3cit (R8) antibody stained only for an extracellular signal at the used concentrations, enabling easier recognition of NETs on the slides. This finding supports the claims from Th&#229;lin et al. that the H3cit (R8) clone can provide a good NET signal with decreased off-target cross-reactivity to non-citrullinated histones28. We also compared MPO antibodies for human and mouse tissue for MPO staining. Our results show that the mouse/human MPO antibody showed more reliable staining and can be used simultaneously on human and mouse samples, simplifying its use in the laboratory. Consequently, we recommend using this antibody combination, H3cit (R8) and mouse/human MPO, for future research and implemented it in our protocol.
However, there is a limitation for multiple staining attempts with this antibody selection. This protocol was modified to use primary antibodies from different hosts. Otherwise, one secondary antibody could bind to both primary antibodies. The H3cit antibody comes from the same host as the NE antibody, so we could not stain NE and H3cit together. Even though no triple staining (NE, H3cit, MPO) was done in this study, this protocol could serve as a foundation for triple-staining experiments in the future. One possible option for triple-staining or NE and H3cit double-staining could be exchanging one of these antibodies with a reliable antibody from a different host. In this case, a possible combination partner could be the NE antibody (Santa Cruz; sc-55549) from sheep used by Knackstedt et al.24. Another option by Duler et al. were published in 2021, where they used a consecutive double-staining method and combined NE and H3cit from a rabbit host26. An additional promising application of a multiple-staining method is for the discrimination between intra- and extravascular NET formation. Instead of triple staining for NET markers, double NET staining could be combined with additional vascular staining. Furthermore, an increasing number of studies have examined the intra- and extravascular distribution of NETs to gain more knowledge on the pathomechanisms of specific diseases, such as Alzheimer&#39;s disease. Smyth et al.31 described a promising and in-depth protocol similar to ours to differentiate between the two possible localizations. They modified their existing NET staining protocol by adding a biotinylated lectin to label endothelial cells and used a fluorophore-conjugated streptavidin for the immunofluorescence staining of lectin. By examining the co-localization of lectin and NET markers in the same image, they were able to identify intravascular NETs31. In this context, further studies are needed to expand the application of our protocol.
After finding the most suitable antibody combination, the next critical step is introducing an autofluorescence-reducing agent. Exogenous factors such as sample fixation using formaldehyde fixatives and endogenous factors such as erythrocytes can result in high autofluorescence, which makes determining co-localization difficult32. This problem mainly occurs in the blue (excitation area: 430-480 nm) and green fluorescence spectra (excitation area: 500-550 nm) and can result in inconclusive staining results when a fluorescence antibody with the same excitation area is used3. The literature reports Sudan Black to be an effective agent for reducing inherent tissue autofluorescence33. Sudan Black makes it possible to obtain clearer staining results using the blue or green fluorescence channels. This excitation area will be necessary for future multiple staining attempts because the blue and green channels are needed when using a fourth fluorescent dye. Our study shows that the optimum incubation time depends on the type of tissue and antibody used in the staining process. Nevertheless, in this work, 5 min of autofluorescence-reducing agent generally gave good results on all tissue types and had the benefit of staining the sample black, thus making it easier to see on the slides. This prevented missing parts of the tissues in the staining process, especially with small samples.
Another essential factor for the success of this protocol is a constant high temperature for the antigen retrieval step. Our research showed that 10 of the 15 previous studies used temperatures above 96 &#176;C for antigen retrieval13,15,16,17,19,21,22,25,26,27. This was consistent with our results, where incubating the samples in a 96 &#176;C water bath or microwave gave good results with no significant differences. Nonetheless, we recommend using a water bath for even heat distribution. When using the microwave, we found a temperature gradient in the buffer of up to 5 &#176;C between the top and the bottom of the staining jar. Additionally, the water bath is easier to use and has a lower risk of the buffer boiling over. For permeabilization, we found that the Triton X-100 had a lesser effect on the staining. So, skipping this step in the protocol is possible without adversely affecting the outcome. Generally, human samples gave better results than mouse samples. A reason for this could be that humans have a higher percentage of neutrophils than mice34,35. Additionally, mouse lung samples showed no visible macroscopic inflammation and fewer neutrophils, while mouse intestine samples were macroscopically inflamed and showed good staining results. Thus, the lower scores in our study could have resulted from the low inflammation and not due to the protocol not working optimally.
For troubleshooting, minor errors in the protocol or not handling the samples carefully may have huge effects on the staining results. One major problem that we identified was sample dehydration. Here, we found that handling more than 10-15 slides at once was critical because every step is time-consuming and can result in dehydration of the samples. Dehydrated samples are no longer usable due to high background staining and no detectable specific fluorescence signal (see Figure 4A). Additionally, the samples should be completely deparaffinized before starting the staining protocol. Paraffin residue resulted in a strong background signal, and the cutting lines could be seen in the stained paraffin (see Figure 4B). Furthermore, after more than 20 freeze-thaw cycles, no signal for the secondary antibody to MPO could be detected (see Figure 4C). Another important step with a great impact on the image quality is the handling of the final mounting step. In this work, air bubbles under the coverslip resulted in the scattering of the fluorescent signal and, thus, a blurry image (see Figure 4D).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65272/65272fig04.jpg" /> 
Figure 4: Troubleshooting tips. (A) This panel shows the consequence of a dried-out sample. The solid red background staining hinders any evaluation of the sample. (B) Here, the red arrow shows paraffin remnants, characterized by the corrugated appearance, after an insufficient deparaffinization. These remnants result in a high background signal and lower-quality images. (C) Inadequate antibody storage or more than 20 freeze-thawing cycles result in the aggregation of the secondary antibody (green arrows) and no binding to MPO. (D) Mounting that is not done carefully can result in air bubbles and, therefore, scattering of the fluorescent light (blue arrow). This can significantly impact the image quality, especially when the scattering blurs the target. For the isotype control, see Supplementary Figure Isocontrol 4. <a href="https://www.jove.com/files/ftp_upload/65272/65272fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Even though NETs are gaining more popularity in scientific research, there are still not enough studies focusing on the methods for NET detection3,13,17,26. To our knowledge, we present the first study comparing protocols from different studies for immunofluorescence imaging of NETs in FFPE tissue. The previously published customized protocols differed in their antibody or antigen retrieval methods and were often designed for only one tissue type, making comparing the NET imaging results more difficult. Therefore, in this study, we identified critical steps and established a protocol suitable for different mouse and human tissues. We achieved this by using a new primary antibody for H3cit staining and reducing the background staining with an autofluorescence-reducing agent. Furthermore, we showed that a constant high temperature is crucial, and careful handling of samples is fundamental for successful NET staining. Therefore, this study provides the essential steps for developing a generally applicable protocol for staining NETs.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65272">Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues</a>. The Authors section was updated from:
Lavinia Sch&#246;enfeld1 
Birgit Appl1 
Laia Pagerols-Raluy1 
Annika Heuer3 
Konrad Reinshagen1 
Michael Boettcher1,2 
1Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf, University of Hamburg 
2Department of Pediatric Surgery, University Medical Center Mannheim, University of Heidelberg 
3Division of Spine Surgery, Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf (UKE)
to:
Lavinia Schoenfeld1 
Birgit Appl1 
Laia Pagerols-Raluy1 
Annika Heuer3 
Konrad Reinshagen1 
Michael Boettcher1,2 
1Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf, University of Hamburg 
2Department of Pediatric Surgery, University Medical Center Mannheim, University of Heidelberg 
3Division of Spine Surgery, Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf (UKE)

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65272">Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues</a>. The Authors section was updated.

Erratum: Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) were first visualized by Brinkmann et al. as a pathway of cellular death different from apoptosis and necrosis in 20041. In this pathway, neutrophils release their decondensed chromatin into the extracellular space to form large web-like structures covered in antimicrobial proteins that were formerly stored in the granules or cytosol. These antimicrobial proteins include neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (H3cit), which are commonly used for indirect immunofluorescence detection of NETs2. This method not only identifies the quantitative presence of these proteins; indeed, it has the advantage of specifically detecting NET-like structures. In the NETs, the mentioned proteins co-localize with extracellular DNA, which can be detected by an overlap of the fluorescence signals of each stained protein and the extracellular DNA. In contrast to the overlapping signals due to extracellular DNA and protein co-localization in NETs, intact neutrophils show no co-localization. Here, the NET components are usually stored separately in the granules, nuclei, and cytosol3.
Since their first discovery, it has been shown that NETs play a central role in numerous diseases, particularly those involving inflammation. NETs show antimicrobial functions during infection through trapping and killing extracellular pathogens in blood and tissue4,5. However, NETs have also been connected to autoimmune diseases and hyperinflammatory responses, like systemic lupus erythematosus, rheumatic arthritis, and allergic asthma6,7,8. NETs promote vaso-occlusion and inflammation in atherosclerosis, platelet adhesion, and are speculated to play a role in metastatic cancer9,10,11. Nevertheless, they are thought to have anti-inflammatory properties by reducing proinflammatory cytokine levels12. While NETs are gaining more interest in a broader field of research, a robust NET detection method is fundamental for future research.
Even though the visualization of NETs in different tissue using immunofluorescence imaging is complex and requires customization, apart from electron microscopy, it is currently one of the most renowned methods for visualizing the interactions between NETs and cells and is predominantly used in formalin-fixed paraffin-embedded tissues (FFPE)13,14. However, comparing NET imaging is difficult, as different laboratories use their own customized protocols. These protocols differ in their use of antibodies, antigen retrieval, or permeabilization method and are often optimized for a specific type of tissue3,13,15,16,17,18,19,20,21,22,23,24,25,26,27.
After Brinkmann et al. published the first methodic study using immunofluorescent visualization of NETs in FFPE tissue, we wanted to optimize this protocol for a wider variety of tissues and species15. Additionally, to establish a broadly applicable immunofluorescence protocol, we tested different modified protocols from studies that used immunofluorescence methods in FFPE tissue to detect NETs3,13,16,17,18,19,20,21,22,23,24,25,26,27. Furthermore, we tried a new H3cit antibody for more specific extracellular staining28. We hypothesize that by systematically adapting current staining protocols to different species and tissue, in vitro imaging can be improved, resulting in a better representation of the interaction between neutrophils and NETs both locally and systemically.

<table><tbody><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&lt;strong&gt;Dilution&lt;/strong&gt;</td></tr><tr><td>Anti-Neutrophil Elastase antibody 100&amp;micro;g</td><td>abcam</td><td>Ab 68672&amp;nbsp;</td><td>1:100</td></tr><tr><td>Anti-Histone H3 (citrulline R2 + R8 + R17) antibody&amp;nbsp; 100&amp;micro;g</td><td>abcam</td><td>Ab 5103</td><td>1:50</td></tr><tr><td>Anti-Myeloperoxidase antibody [2C7] anti-human 100 &amp;micro;g</td><td>abcam</td><td>Ab 25989</td><td>1:50</td></tr><tr><td>Anti-Myeloperoxidase antibody [2D4] anti-mouse 50 &amp;micro;g</td><td>abcam</td><td>Ab 90810</td><td>1:50</td></tr><tr><td>Axiovision Microscopy Software&amp;nbsp;</td><td>Zeiss</td><td>4.8.2.</td><td></td></tr><tr><td>Blocking solution with donkey serum (READY TO USE) 50ml</td><td>GeneTex</td><td>&amp;nbsp;GTX30972</td><td></td></tr><tr><td>Coverslips</td><td>Marienfeld</td><td>0101202</td><td></td></tr><tr><td>Dako Target Retrieval Solution Citrate pH6 (x10)</td><td>Dako</td><td>S2369</td><td></td></tr><tr><td>DAPI 25 mg</td><td>Roth</td><td>6335.1</td><td>1:25000</td></tr><tr><td>DCS antibody dilution 500 mL</td><td>DCS diagnostics</td><td>DCS AL120R500</td><td></td></tr><tr><td>Donkey ant goat Cy3</td><td>JacksonImmunoResearch</td><td>705-165-147</td><td>1:200</td></tr><tr><td>Donkey anti rabbit AF647</td><td>JacksonImmunoResearch</td><td>711-605-152</td><td>1:200</td></tr><tr><td>Donkey anti rabbit Cy3</td><td>JacksonImmunoResearch</td><td>711-165-152</td><td>1:200</td></tr><tr><td>Fluoromount-G Mounting Medium</td><td>Invitrogen</td><td>00-4958-02</td><td></td></tr><tr><td>Glass slide rack</td><td>Roth</td><td>H552.1</td><td></td></tr><tr><td>Human/Mouse MPO Antibody</td><td>R&amp;amp;D Systems</td><td>AF 3667&amp;nbsp;</td><td>1:20</td></tr><tr><td>Hydrophobic Pen</td><td>KISKER</td><td>MKP-1</td><td></td></tr><tr><td>Isokontrolle Rabbit IgG Polyclonal 5mg</td><td>abcam</td><td>Ab 37415</td><td>1:2000 and 1:250</td></tr><tr><td>MaxBlock Autofluorescence Reducing Reagent Kit (RUO) 100 ml</td><td>Maxvision</td><td>MB-L</td><td></td></tr><tr><td>Microscopy camera</td><td>Zeiss</td><td>AxioCamHR3</td><td></td></tr><tr><td>Microwave</td><td>Bosch</td><td>HMT84M421</td><td></td></tr><tr><td>Mouse IgG1 negative control</td><td>Dako</td><td>X0931 Aglient</td><td>1:50 and 1:5</td></tr><tr><td>Normal Goat IgG Control</td><td>R&amp;amp;D Systems</td><td>AB-108-C&amp;nbsp;</td><td>1:100</td></tr><tr><td>PBS Phosphate buffered saline (10x)</td><td>Sigma-Aldrich</td><td>P-3813</td><td></td></tr><tr><td>PMP staining jar</td><td>Roth</td><td>2292.2</td><td></td></tr><tr><td>Recombinant Anti-Histone H3 (citrulline R8) antibody 100&amp;micro;g</td><td>abcam</td><td>Ab 219406</td><td>1:100</td></tr><tr><td>Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control 200&amp;micro;g</td><td>abcam</td><td>Ab 172730</td><td>1:300</td></tr><tr><td>ROTI Histol</td><td>Roth</td><td>&amp;nbsp;6640</td><td></td></tr><tr><td>SuperFrost Plus slides</td><td>R. Langenbrinck</td><td>03-0060</td><td></td></tr><tr><td>TBS Tris buffered saline (x10)</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>Water bath</td><td>Memmert</td><td>830476</td><td></td></tr><tr><td>Water bath rice cooker</td><td>reishunger</td><td>RCP-30</td><td></td></tr><tr><td>Wet chamber</td><td>Weckert Labortechnik</td><td>600016</td><td></td></tr><tr><td>Zeiss Widefield microscope</td><td>Zeiss</td><td>Axiovert 200M</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues at JoVE.com

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.

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 ...

immunofluorescence-imaging-neutrophil-extracellular-traps-human-mouse

North Carolina State University

Research

JoVE Journal

Immunology and Infection

6.2K Views.  University of Hamburg. 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. 

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

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 ...

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 ...

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.

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 ...

Medicine

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive Interaction Between Cancer Cells and NETs

Source: Kanamaru, R. et al. <a href="https://www.jove.com/video/58201" target="_blank">Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment.</a> J. Vis. Exp. (2018)
This video demonstrates the fluorescent staining of neutrophil extracellular traps (NETs), the extracellular DNA components expelled by neutrophils. This technique enables the visualization of adhesive interaction between cancer cells and NETs.

Source: Kanamaru, R. et al. Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell ...

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

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

Source: Arends, E. J. et al., <a href="https://app.jove.com/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 Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Summary

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

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

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive Interaction Between Cancer Cells and NETs

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