这项研究由德国研究学会（BO5534）创立。我们感谢 Antonia Kiwitt、Moritz Lenz、Johanna Hagens、Annika Heuer 博士和 PD Ingo Königs 博士为我们提供样品。此外，作者还感谢UKE显微镜成像设施（UKE医学院核心设施）的团队对免疫荧光显微镜的支持。

这项研究包括来自汉堡国家动物研究局批准的实验的小鼠组织，Behörde für Justiz und Verbraucherschutz，汉堡，德国（73/17,100/17,94/16,109/2018,63/16）。使用的组织是来自化粪池模型的小鼠肺和结肠以及烧伤的皮肤。我们使用了 8 周龄的雄性和雌性小鼠。所有实验均遵循关于保护用于科学目的的动物的欧洲指令 2010/63/EU。匿名的人类样本包括新生儿小肠结肠炎、烧伤皮肤、胆道闭锁、椎间盘炎和心肌的组织。根据汉堡医学研究伦理委员会的说法，这些样本不需要知情同意，但该研究得到了委员会的批准（WF-026/21）。
1. 样品固定
<ol><li>使用源自 Abu Abed 和 Brinkmann 的以下方案进行样品固定、脱水、石蜡包埋、切片和封片 3,15。<ol><li>通过将 40 g 多聚甲醛 （PFA） 溶解在 800 mL Tris 缓冲盐水 （pH 7.4） （TBS） 中来制备 4% 甲醛溶液。</li>
		<li>在通风橱下在60°C下搅拌混合物，直到PFA溶解。将溶液置于室温 （RT），并用 TBS 将体积调节至 1,000 mL。</li>
		<li>将pH值调节至7.4。在4°C下储存2-3周或在-20°C下储存长达1年。</li>
	</ol></li>
	<li>对于样品固定，将新鲜组织放入TBS缓冲液中，并解剖成小于20 mm x 30 mm x 3 mm的碎片。将组织切片浸入4%多聚甲醛溶液中12-24小时。 注：原始方案使用2%PFA溶液，固定时间为8-20小时。使用这种方法，一些组织切片在20小时后没有完全固定，因此PFA浓度增加到4%PFA。</li>
	<li>将样品转移到标记的组织处理盒中。使用耐溶剂记号笔或铅笔进行标记。</li>
	<li>通过将盒浸入70%乙醇中1小时开始样品脱水。然后，浸入80%乙醇，90%乙醇，96%乙醇中，并在100%乙醇中浸泡两次，每步持续1小时。</li>
	<li>在100%二甲苯（二甲苯）中浸泡两次，然后在60°C石蜡中浸泡两次，每次浸泡1小时。</li>
	<li>使用嵌入模具进行安装，并在取出模具之前让石蜡固化。</li>
	<li>使用切片机切割3μm厚的组织切片。</li>
	<li>用镊子将切割部分铺设在37°C的水浴中，让它们漂浮在表面上以拉伸组织切割。</li>
	<li>将切片放在粘性载玻片上（材料表），并在40°C加热室中干燥过夜。</li>
</ol>2. 样品补水
<ol><li>对于脱蜡，将载玻片堆叠在载玻片架中，并在二甲苯替代介质柠檬烯（材料表）中浸没两次，每次浸没5分钟，在1：1柠檬烯/乙醇混合物中浸泡一次5分钟。 注意：柠檬烯在50°C时易燃，会引起过敏反应。在通风橱下使用，戴上护目镜和手套。远离明火。</li>
	<li>通过将载玻片架浸入100%乙醇中两次，一次浸入96%乙醇，90%乙醇，80%乙醇和70%乙醇中，每次浸泡5分钟，使样品在下降乙醇系列中再水化。</li>
	<li>将载玻片架浸入去离子水（去离子水）中5分钟，以清除任何残留的乙醇。</li>
</ol>3. 自发荧光阻断和抗原修复
<ol><li>根据数据表制备pH 6柠檬酸盐目标修复溶液（TRS）（材料表）。该TRS浓缩10倍。因此，用去离子水按 1：10 稀释。在塑料染色罐中预热至96°C。 注：TRS破坏样品固定过程中形成的亚甲基桥，暴露抗原表位。这使得一抗能够结合其靶抗原。将浓缩的TRS在2-8°C下储存8个月。始终准备新鲜的 TRS。</li>
	<li>将载玻片从载玻片架中取出，然后将它们放入湿室中。将一到两滴自发荧光还原剂（材料表）移液到每个样品上。孵育 5 分钟。 注：自发荧光还原剂可阻断由内源性荧光团和固定剂引起的固有组织自发荧光。将自发荧光还原剂在室温下储存长达 1 年。 注意：仅处理小块组织，以避免样品变干或超过孵育时间。</li>
	<li>将载玻片堆叠回载玻片架中，并在60%乙醇中以向上和向下的动作冲洗1分钟，直到所有未使用的自发荧光还原试剂被洗掉。</li>
	<li>将载玻片架浸入去离子水中5分钟。</li>
	<li>为了抗原修复，将载玻片转移到带有预热 TRS 的染色罐中。在96°C的水浴中孵育10分钟。 注意：如果将TRS预热至96°C并确保在96°C下孵育10分钟，则可以用微波炉或蒸汽锅代替96°C水浴。</li>
	<li>取出染色罐，让它慢慢冷却至室温约60-90分钟。 注意：该协议可以在此处暂停长达4小时，将载玻片留在TRS中。</li>
	<li>取出 TRS，但将载玻片放在罐子里。用含有0.05%吐温（TBST，pH 7.4）的Tris缓冲盐水冲洗两次，每次3分钟，以洗去任何剩余的TRS残留物。</li>
	<li>对于透化，在磷酸盐缓冲盐水（PBS）中制备0.2%Triton，pH 7.4，并将其填充到染色罐中以透化样品10分钟。 注：透化可增强一抗与固定样品中细胞内靶蛋白的结合。</li>
	<li>在TBST中再次冲洗载玻片两次，每次3分钟。</li>
	<li>准备湿室，并放置载玻片。用纸巾擦去载玻片上多余的水。用疏水屏障笔圈出每个样品，以防止抗体溶液流失。 注意： 不要让样品变干。最好使用小部分。</li>
</ol>4. 阻断非特异性抗体结合
<ol><li>取含有驴血清的即用型封闭液（材料表），吸取一滴到每个样品上。在室温下孵育 30 分钟。 注：此封闭步骤可最大限度地减少二抗与样品上非特异性结合位点的结合。在阻断这些非特异性表位并应用一抗后，二抗将与一抗结合，不与组织表面相互作用。这种即用型封闭试剂在2-8°C下储存6个月。</li>
	<li>将载玻片的边缘轻敲坚硬的表面，去除载玻片上多余的堵塞溶液。不要冲洗它们。</li>
</ol>5. 一抗
<ol><li>在抗体稀释缓冲液中稀释一抗（材料表）。每个样品使用大约 100 μL 的最终溶液。对于 NE 和 H3cit （R8） 抗体和同质对照，稀释至 5 μg/mL 的浓度。对于MPO和相应的isocontrol，使用10μg/ mL。为每种一抗准备一管，为每种同对照抗体准备一管。 注意：H3cit （R8）/MPO 或 NE/MPO 及其等对照可以组合用于 NET 染色。对于 H3cit （R8）/MPO 的组合，将 H3cit （R8） 的终浓度稀释至 5 μg/mL，将 MPO 的终浓度稀释至 10 μg/mL，至每个样品的终体积为 100 μL。对于 NE/MPO，将 NE 的终浓度稀释至 5 μg/mL，MPO 的终浓度为 10 μg/mL，每个样品的终体积为 100 μL。对于isocontrol，使用与每种相应抗体相同的浓度。始终对使用的每种抗体使用新的移液器吸头。一抗可在 4 °C 下储存 1-2 周，在 -20 °C 或 -80 °C 下储存长达 1 年。</li>
	<li>在一张载玻片上放置两个样品，一个用于 isocontrol 抗体，另一个用于 MPO/H3cit 或 MPO/NE 抗体稀释。每个样品使用约 100 μL，并将抗体溶液均匀分布。 注意： 不要让样品变干。注意避免混淆同对照抗体和一抗。</li>
	<li>在4°C的湿室中储存过夜。</li>
	<li>第二天，从载玻片中取出多余的一抗溶液，然后将载玻片堆叠在比色皿中。用TBST冲洗3次，每次5分钟，以洗去剩余的一抗溶液。</li>
</ol>6. 二抗
<ol><li>制备二抗溶液。使用两种不同的荧光二抗：驴-抗山羊用于 MPO 和驴-抗兔用于 H3cit 染色。用抗体稀释缓冲液将每种稀释液稀释至7.5μg/ mL的浓度（材料表）。 注：对于双染色，使用两种具有不同激发区域和最小光谱重叠的荧光抗体。合并两种二抗，并将每种抗体的终浓度稀释至 7.5 μg/mL，使每个样品的最终体积为 100 μL。保护抗体避光。二抗可在 2-8 °C 下储存 6-8 周，或在 -80 °C 下储存长达 1 年。</li>
	<li>将载玻片保持在湿室中，并在每个样品上加入 100 μL 二抗溶液。让它们在室温下孵育 30 分钟并避光。</li>
	<li>轻拍多余的二抗溶液，然后将载玻片堆叠在载玻片架中。在装有 PBS 的染色罐中浸没 3 次，每次 5 分钟，以洗去任何剩余的未结合抗体。</li>
	<li>制备DAPI（4'，6-二脒基-2-苯基吲哚）溶液（材料表），并用去离子水稀释至1μg/ mL的浓度。将载玻片架浸入装有DAPI溶液的染色罐中，并在室温下在黑暗中孵育5分钟。 注：DAPI用作双链DNA的荧光染料。制备的DAPI溶液可以多次使用。将制备好的DAPI溶液储存在室温下。DAPI浓缩液可在-20°C下储存长达1年。</li>
	<li>将载玻片架浸入装有PBS的染色罐中5分钟，以洗去多余的DAPI溶液。</li>
</ol>7. 安装和储存样品，显微镜分析
<ol><li>用盖玻片和安装介质（材料表）安装样品。 注意： 仅使用少量封固剂，并用湿纸巾擦去多余的介质。戴上手套，避免意外擦掉盖玻片或载玻片上的任何介质。避免气泡形成，并用棉签轻轻按压盖玻片，以去除载玻片上的任何气泡。</li>
	<li>对于成像，请使用宽视场显微镜或共聚焦显微镜。 注意： 首先拍摄 isocontrol 的图像。降低荧光滤光片的曝光时间（DAPI的蓝色滤光片除外），直到几乎无法检测到信号。然后，使用此设置检查相应的样本。使用荧光通道寻找可以在每个样品中检测到荧光信号的区域。在拍摄该区域的图像后，程序会生成所有通道的复合图像，并将不同的荧光信号显示为不同颜色的重叠。然后可以进一步分析图像，以便与成像软件（如 ImageJ）进行共定位。</li>
	<li>将载玻片在4°C下储存长达6个月。</li>
</ol>

染色和成像以可视化中性粒细胞胞外陷阱

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M., 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=Renal+participation+of+myeloperoxidase+in+antineutrophil+cytoplasmic+antibody+(ANCA)-associated+glomerulonephritis.">Renal participation of myeloperoxidase in antineutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis.</a> Kidney International. 88 (5), 1030-1046 (2015).</li><li>Barliya, 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=Possible+involvement+of+NETosis+in+inflammatory+processes+in+the+eye:+Evidence+from+a+small+cohort+of+patients.">Possible involvement of NETosis in inflammatory processes in the eye: Evidence from a small cohort of patients.</a> Molecular Vision. 23, 922-932 (2017).</li><li>Xu, D., 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=Overproduced+bone+marrow+neutrophils+in+collagen-induced+arthritis+are+primed+for+NETosis:+An+ignored+pathological+cell+involving+inflammatory+arthritis.">Overproduced bone marrow neutrophils in collagen-induced arthritis are primed for NETosis: An ignored pathological cell involving inflammatory arthritis.</a> Cell Proliferation. 53 (7), e12824 (2020).</li><li>Nonokawa, M., 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=Association+of+neutrophil+extracellular+traps+with+the+development+of+idiopathic+osteonecrosis+of+the+femoral+head.">Association of neutrophil extracellular traps with the development of idiopathic osteonecrosis of the femoral head.</a> American Journal of Pathology. 190 (11), 2282-2289 (2020).</li><li>Tucker, S. L., Sarr, D., Rada, B. <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+are+present+in+the+airways+of+ENaC-overexpressing+mice+with+cystic+fibrosis-like+lung+disease.">Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease.</a> BMC Immunology. 22 (1), 7 (2021).</li><li>Knackstedt, S. L., 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+drive+inflammatory+pathogenesis+in+malaria.">Neutrophil extracellular traps drive inflammatory pathogenesis in malaria.</a> Science Immunology. 4 (40), (2019).</li><li>Nakazawa, D., 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=Histones+and+neutrophil+extracellular+traps+enhance+tubular+necrosis+and+remote+organ+injury+in+ischemic+AKI.">Histones and neutrophil extracellular traps enhance tubular necrosis and remote organ injury in ischemic AKI.</a> Journal of the American Society of Nephrology. 28 (6), 1753-1768 (2017).</li><li>Duler, L., Nguyen, N., Ontiveros, E., Li, R. H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neutrophil+extracellular+traps+in+paraffin-embedded+feline+arterial+thrombi+using+immunofluorescence+microscopy.">Identification of neutrophil extracellular traps in paraffin-embedded feline arterial thrombi using immunofluorescence microscopy.</a> Journal of Visualized Experiments. (157), e60834 (2020).</li><li>Stehr, A. M., 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+drive+epithelial-mesenchymal+transition+of+human+colon+cancer.">Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer.</a> Journal of Pathology. 256 (4), 455-467 (2022).</li><li>Th&#229;lin, C., 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=Quantification+of+citrullinated+histones:+Development+of+an+improved+assay+to+reliably+quantify+nucleosomal+H3Cit+in+human+plasma.">Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.</a> Journal of Thrombosis and Haemostasis. 18 (10), 2732-2743 (2020).</li><li>Yamashita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval:+Mechanisms+and+application+to+histochemistry.">Heat-induced antigen retrieval: Mechanisms and application to histochemistry.</a> Progress in Histochemistry and Cytochemistry. 41 (3), 141-200 (2007).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+cell+membranes.">Permeabilization of cell membranes.</a> Methods in Molecular Biology. 588, 63-66 (2010).</li><li>Smyth, L., 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-vascular+interactions+drive+myeloperoxidase+accumulation+in+the+brain+in+Alzheimer's+disease.">Neutrophil-vascular interactions drive myeloperoxidase accumulation in the brain in Alzheimer's disease.</a> Acta Neuropathologica Communications. 10 (1), 38 (2022).</li><li>Whittington, N. C., Wray, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+red+blood+cell+autofluorescence+for+immunocytochemistry+on+fixed+embryonic+mouse+tissue.">Suppression of red blood cell autofluorescence for immunocytochemistry on fixed embryonic mouse tissue.</a> Current Protocols in Neuroscience. 81, 2-22 (2017).</li><li>Nazir, S., Charlesworth, R. P. G., Moens, P., Gerber, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+autofluorescence+quenching+techniques+on+formalin-fixed+chicken+tissues.">Evaluation of autofluorescence quenching techniques on formalin-fixed chicken tissues.</a> Journal of Immunological Methods. 496, 113097 (2021).</li><li>Schneider, C., 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=IVIG+regulates+the+survival+of+human+but+not+mouse+neutrophils.">IVIG regulates the survival of human but not mouse neutrophils.</a> Scientific Reports. 7 (1), 1296 (2017).</li><li>Risso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+antimicrobial+peptides:+Multifunctional+effector+molecules+of+innate+immunity.">Leukocyte antimicrobial peptides: Multifunctional effector molecules of innate immunity.</a> Journal of Leukocyte Biology. 68 (6), 785-792 (2000).</li></ol>

作者没有什么可透露的。

在这项工作中，我们旨在从实际染色过程开始，调整和优化现有的NET成像方案，以适应更多的组织类型。该方法的第一个关键步骤是选择最合适的抗体。对于NE，我们在人体组织上尝试了来自小鼠宿主的NE抗体，与来自兔宿主的NE相比，该抗体没有显示出可靠的染色。此外，Thålin 等人提出 H3cit （R8） 作为细胞外染色的更具特异性的抗体。我们将该抗体与广泛使用的triH3cit（R2，R8，R17）进行了比较?...

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

Brinkmann 等人于 2004 年首次将中性粒细胞胞外陷阱 （NET） 可视化为不同于细胞凋亡和坏死的细胞死亡途径1。在该途径中，中性粒细胞将其解凝的染色质释放到细胞外空间，形成覆盖着先前储存在颗粒或胞质溶胶中的抗菌蛋白的大型网状结构。这些抗菌蛋白包括中性粒细胞弹性蛋白酶 （NE）、髓过氧化物酶 （MPO） 和瓜氨酸组蛋白 H3 （H3cit），它们通常用于 NET 的间接免疫荧光检测2。该方法不仅可以鉴定这些蛋白质的定量存在;事实上，它具有专门检测类NET结构的优点。在NET中，上述蛋白质与细胞外DNA共定位，这可以通过每个染色蛋白质和细胞外DNA的荧光信号的重叠来检测。与NET中细胞外DNA和蛋白质共定位引起的重叠信号相反，完整的中性粒细胞没有共定位。在这里，NET 组分通常分别储存在颗粒、细胞核和胞质溶胶3 中。
自首次发现以来，已经表明NET在许多疾病中起着核心作用，特别是那些涉及炎症的疾病。NETs在感染期间通过捕获和杀死血液和组织中的细胞外病原体显示出抗菌功能4,5。然而，NET也与自身免疫性疾病和过度炎症反应有关，如系统性红斑狼疮、风湿性关节炎和过敏性哮喘6,7,8。NETs促进动脉粥样硬化中的血管闭塞和炎症，血小板粘附，并被推测在转移性癌症中发挥作用9,10,11。然而，它们被认为通过降低促炎细胞因子水平而具有抗炎特性12。虽然神经内分泌瘤在更广泛的研究领域越来越受到关注，但强大的神经内分泌瘤检测方法是未来研究的基础。
尽管使用免疫荧光成像对不同组织中的NET进行可视化很复杂并且需要定制，但除了电子显微镜之外，它是目前最著名的可视化NET与细胞之间相互作用的方法之一，主要用于福尔马林固定石蜡包埋组织（FFPE）13,14.然而，比较NET成像是困难的，因为不同的实验室使用自己的定制方案。这些方案在抗体、抗原修复或透化方法的使用上有所不同，并且通常针对特定类型的组织进行优化 3,13,15,16,17,18,19,20,21,22,23,24,25,26 ，27.
在 Brinkmann 等人发表了第一项使用免疫荧光可视化 FFPE 组织中 NET 的方法研究后，我们希望针对更广泛的组织和物种优化该协议15。此外，为了建立广泛适用的免疫荧光方案，我们测试了在FFPE组织中使用免疫荧光方法检测NETs 3,13,16,17,18,19,20,21,22,23,24,25，26,27.此外，我们尝试了一种新的 H3cit 抗体用于更特异性的细胞外染色28。我们假设，通过系统地使当前的染色方案适应不同的物种和组织，可以改善体外成像，从而更好地表示局部和全身中性粒细胞和NET之间的相互作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Anti-Neutrophil Elastase antibody 100&#181;g</td>
			<td>abcam</td>
			<td>Ab 68672&#160;</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Anti-Histone H3 (citrulline R2 + R8 + R17) antibody&#160; 100&#181;g</td>
			<td>abcam</td>
			<td>Ab 5103</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Anti-Myeloperoxidase antibody [2C7] anti-human 100 &#181;g</td>
			<td>abcam</td>
			<td>Ab 25989</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Anti-Myeloperoxidase antibody [2D4] anti-mouse 50 &#181;g</td>
			<td>abcam</td>
			<td>Ab 90810</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Axiovision Microscopy Software&#160;</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>&#160;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&#38;D Systems</td>
			<td>AF 3667&#160;</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&#38;D Systems</td>
			<td>AB-108-C&#160;</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&#181;g</td>
			<td>abcam</td>
			<td>Ab 219406</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control 200&#181;g</td>
			<td>abcam</td>
			<td>Ab 172730</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>ROTI Histol</td>
			<td>Roth</td>
			<td>&#160;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

中性粒细胞胞外陷阱 （NET） 由中性粒细胞释放，作为对细菌感染或创伤性组织损伤的反应，但也在自身免疫性疾病和无菌性炎症中发挥作用。它们是由双链 DNA 细丝、组蛋白和抗菌蛋白组成的网状结构。一旦释放，NETs可以捕获并杀死血液和组织中的细胞外病原体。此外，NET通过刺激血小板粘附和凝血来参与稳态调节。然而，神经内分泌失调的产生也与各种疾病有关，包括败血症或自身免疫性疾病，这使它们成为治疗干预的有希望的靶点。除电子显微镜外，使用免疫荧光成像可视化NET是目前唯一已知的证明组织中NET相互作用的方法之一。因此，已经使用了各种染色方法来可视化 NET。在文献中，描述了不同的染色方案，我们确定了方案之间显示出高度变异性的四个关键组成部分：（1）使用的抗体类型，（2）自发荧光还原剂的使用，（3）抗原修复方法，以及（4）透化。因此，在这项工作中系统地调整和改进 了体外 免疫荧光染色方案，使其适用于不同的物种（小鼠、人类）和组织（皮肤、肠道、肺、肝脏、心脏、椎间盘）。固定和石蜡包埋后，将 3 μm 厚的切片安装到载玻片上。根据改良的染色方案，用髓过氧化物酶 （MPO）、瓜氨酸组蛋白 H3 （H3cit） 和中性粒细胞弹性蛋白酶 （NE） 的一抗对这些样品进行染色。用二抗对载玻片进行染色，并使用宽场荧光显微镜进行检查。根据评估表对结果进行分析，并半定量记录差异。
在这里，我们提出了一种适用于不同组织的优化NET染色方案。我们使用一种新型一抗对 H3cit 进行染色，并使用自发荧光还原剂减少非特异性染色。此外，我们证明了NET染色需要恒定的高温和小心处理样品。

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.

作者聚焦：人和小鼠组织中的中性粒细胞胞外陷阱成像 

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Among antibody selection for tissue imaging, the fixation process poses a challenge by epitope masking. Therefore, customized retriever step is required to expose these antigens for antibody binding. Additionally, tissue inherent autofluorescence can cause difficulties through to a high background staining.However, the use of different approaches in the protocols in the literature hinders a standardized comparison between the images. Our study aims to address the demand for standardized procedures and assist the researcher in overcoming challenges during the imaging process. We established a versatile imaging protocol applicable to different tissues by comparing various different protocols.So our work provides guiding and troubleshooting tips from antibody usage to sample mounting, highlighting the potential pitfalls. Our findings advance research by introducing a new primary antibody for citrullinated hisone three, and an autofluorescent reducing agent to minimize background staining. Furthermore, for successful imaging, we emphasize the careful handling of samples and the importance of maintaining a constant high temperature for antigen retrieval.

在开始优化方案之前，我们通过在 PubMed 上搜索使用 FFPE 组织对 NET 进行免疫染色的研究并比较其方案，确定了成功染色的关键步骤。最有希望的协议差异被确定为协议优化的关键步骤，而大部分相互对应的步骤没有改变（表1）。
表 1：用于 NET FFPE 免疫染色的 PubMed 研究。 该表显示了所检查研究中免疫染色方案中的变量。所使用的协议被划分为它们?...

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

中性粒细胞胞外陷阱 （NET） 与多种疾病有关，免疫荧光通常用于其可视化。然而，有各种染色方案，在许多情况下，只检查一种类型的组织。在这里，我们建立了一种普遍适用的方案，用于在小鼠和人体组织中染色NET。

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

在用于组织成像的抗体选择中，固定过程对表位掩蔽提出了挑战。因此，需要定制的回收器步骤来暴露这些抗原以进行抗体结合。此外，组织固有的自发荧光会导致高背景染色困难。然而，在文献中的协议中使用不同的方法阻碍了图像之间的标准化比较。我们的研究旨在满足对标准化程序的需求，并帮助研究人员克服成像过程中的挑战。通过比较各种不同的方案，我们建立了适用于不同组织的多功能成像方案。因此，我们的工作提供了从抗体使用到样品安装的指导和故障排除技巧，突出了潜在的陷阱。我们的研究结果通过引入一种新的瓜氨酸化苏松三一抗和一种自发荧光还原剂来最大限度地减少背景染色，从而推进了研究。此外，为了成功成像，我们强调对样本的小心处理以及保持恒定高温以进行抗原修复的重要性。

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Research

JoVE Journal

Immunology and Infection

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

3.9K 次观看.  University of Hamburg. 在用于组织成像的抗体选择中，固定过程对表位掩蔽提出了挑战。因此，需要定制的回收器步骤来暴露这些抗原以进行抗体结合。此外，组织固有的自发荧光会导致高背景染色困难。然而，在文献中的协议中使用不同的方法阻碍了图像之间的标准化比较。我们的研究旨在满足对标准化程序的需求，并帮助研究人员克服成像过程中的挑战。通过比较各种不同的方案，我们?...