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 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 µm 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.
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.
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, Thålin et al. proposed H3cit (R8) as a more specific antibody for extracellular staining. We compared this antibody with the widely…
The authors have nothing to disclose.
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 Königs 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.
Dilution | |||
Anti-Neutrophil Elastase antibody 100µg | abcam | Ab 68672 | 1:100 |
Anti-Histone H3 (citrulline R2 + R8 + R17) antibody 100µg | abcam | Ab 5103 | 1:50 |
Anti-Myeloperoxidase antibody [2C7] anti-human 100 µg | abcam | Ab 25989 | 1:50 |
Anti-Myeloperoxidase antibody [2D4] anti-mouse 50 µg | abcam | Ab 90810 | 1:50 |
Axiovision Microscopy Software | Zeiss | 4.8.2. | |
Blocking solution with donkey serum (READY TO USE) 50ml | GeneTex | GTX30972 | |
Coverslips | Marienfeld | 0101202 | |
Dako Target Retrieval Solution Citrate pH6 (x10) | Dako | S2369 | |
DAPI 25 mg | Roth | 6335.1 | 1:25000 |
DCS antibody dilution 500 mL | DCS diagnostics | DCS AL120R500 | |
Donkey ant goat Cy3 | JacksonImmunoResearch | 705-165-147 | 1:200 |
Donkey anti rabbit AF647 | JacksonImmunoResearch | 711-605-152 | 1:200 |
Donkey anti rabbit Cy3 | JacksonImmunoResearch | 711-165-152 | 1:200 |
Fluoromount-G Mounting Medium | Invitrogen | 00-4958-02 | |
Glass slide rack | Roth | H552.1 | |
Human/Mouse MPO Antibody | R&D Systems | AF 3667 | 1:20 |
Hydrophobic Pen | KISKER | MKP-1 | |
Isokontrolle Rabbit IgG Polyclonal 5mg | abcam | Ab 37415 | 1:2000 and 1:250 |
MaxBlock Autofluorescence Reducing Reagent Kit (RUO) 100 ml | Maxvision | MB-L | |
Microscopy camera | Zeiss | AxioCamHR3 | |
Microwave | Bosch | HMT84M421 | |
Mouse IgG1 negative control | Dako | X0931 Aglient | 1:50 and 1:5 |
Normal Goat IgG Control | R&D Systems | AB-108-C | 1:100 |
PBS Phosphate buffered saline (10x) | Sigma-Aldrich | P-3813 | |
PMP staining jar | Roth | 2292.2 | |
Recombinant Anti-Histone H3 (citrulline R8) antibody 100µg | abcam | Ab 219406 | 1:100 |
Recombinant Rabbit IgG, monoclonal [EPR25A] – Isotype Control 200µg | abcam | Ab 172730 | 1:300 |
ROTI Histol | Roth | 6640 | |
SuperFrost Plus slides | R. Langenbrinck | 03-0060 | |
TBS Tris buffered saline (x10) | Sigma-Aldrich | T1503 | |
Triton X-100 | Sigma-Aldrich | T8787 | |
Tween 20 | Sigma-Aldrich | P9416 | |
Water bath | Memmert | 830476 | |
Water bath rice cooker | reishunger | RCP-30 | |
Wet chamber | Weckert Labortechnik | 600016 | |
Zeiss Widefield microscope | Zeiss | Axiovert 200M |