Name: identification of neutrophil extracellular traps in paraffin-embedded feline arterial thrombi using immunofluorescence microscopy
Uploaded: 2020-03-29T13:00:00
Description: Watch this Scientific Journal Video about Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy at JoVE.com

这项研究得到了加州大学戴维斯分校动物健康中心（CCAH 2018-30-F）的资金支持。作者要感谢凯文·伍拉德博士使用荧光显微镜。

此处描述的所有方法均按照加州大学戴维斯分校机构动物护理和使用委员会的准则执行。在业主同意下进行组织解剖和活检。
1. 组织固定、嵌入和切片
<ol><li>在人道安乐死或死亡后不久，切除主动脉分叉，包括下降的主动脉、股动脉和常见的伊利亚动脉（图1A）。布伦特解剖出筋膜 （图 1B） 之前，它完全浸入 10% 中性缓冲形式，...

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H. L., Tablin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Canine+Neutrophil+Extracellular+Trap+Formation:+Dynamic+and+Quantitative+Analysis+by+Fluorescence+Microscopy.">In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy.</a> Journal of Visualized Experiments. (138), e58083 (2018).</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>Farkas, &. #. 1. 9. 3. ;. Z., 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+in+thrombi+retrieved+during+interventional+treatment+of+ischemic+arterial+diseases.">Neutrophil extracellular traps in thrombi retrieved during interventional treatment of ischemic arterial diseases.</a> Thrombosis Research. 175, 46-52 (2019).</li><li>Qi, H., Yang, S., Zhang, L. <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+and+Endothelial+Dysfunction+in+Atherosclerosis+and+Thrombosis.">Neutrophil Extracellular Traps and Endothelial Dysfunction in Atherosclerosis and Thrombosis.</a> Frontiers in Immunology. 8, 928 (2017).</li><li>Laridan, E., 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+in+ischemic+stroke+thrombi.">Neutrophil extracellular traps in ischemic stroke thrombi.</a> Annals of Neurology. 82 (2), 223-232 (2017).</li><li>Laridan, E., Martinod, K., Meyer, S. F. D. <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+Arterial+and+Venous+Thrombosis.">Neutrophil Extracellular Traps in Arterial and Venous Thrombosis.</a> Seminars in Thrombosis and Hemostasis. 45 (1), 86-93 (2019).</li><li>Li, R. H. L., Johnson, L. R., Kohen, C., Tablin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+identifying+and+quantifying+neutrophil+extracellular+trap+formation+in+septic+dogs+using+immunofluorescence+microscopy.">A novel approach to identifying and quantifying neutrophil extracellular trap formation in septic dogs using immunofluorescence microscopy.</a> BMC Veterinary Research. 14 (1), 210 (2018).</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>Moelans, C. B., Oostenrijk, D., Moons, M. J., van Diest, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+substitute+fixatives:+effects+on+nucleic+acid+preservation.">Formaldehyde substitute fixatives: effects on nucleic acid preservation.</a> Journal of Clinical Pathology. 64 (11), 960-967 (2011).</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: A Journal of Technical Methods and Pathology. 84 (3), 300-306 (2004).</li><li>Willingham, M. 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我们描述了一种协议，使用双免疫标记协议和免疫荧光显微镜识别固定猫科动物心电源动脉血栓中的NET。虽然只有心电动脉血栓被染色，但理论上，这种协议可用于其他类型的血栓和其他兽医物种。猫动脉血栓内NET的识别表明，NET在猫的血栓形成中可能起一定的作用。
在固定和石蜡嵌入组织中通过免疫荧光检测NET优于H&E等传统组织学污渍，后者通常显示由嗜中性粒细胞包围?...

患有肥大心肌病的猫有生命危险血栓栓塞并发症11，2。2尽管与猫科动物心电源性动脉血栓栓塞（CATE）相关的发病率和死亡率很高，但CAT在猫身上的基本病理生理学还知之甚少。也有有限的诊断和治疗工具，以治疗和识别猫在这种毁灭性的条件3的风险。
除了在先天免疫中的作用外，中性粒细胞外嗜中性粒细胞外陷阱（NETs）在血栓形成中发挥了作用，这种陷阱是网络状的无细胞DNA（cfDNA）网络，含有组蛋白和粒状蛋白质，如中性粒细胞酶酶（NE）和骨髓红酶。嗜中性粒细胞在反应全身炎症、直接接触病原体以及与活性血小板44、5、6、75,6,7相互作用时进行神经衰弱的形成。在狗中，中性粒细胞衍生的DNA已被证明能抑制血块性结质，而NET蛋白加速血块形成。NET捕获循环细胞和凝固成分的能力也是其血栓生成特性88、9、10、11、12的关键。,9,10,11,12
通过细胞外嗜中性粒细胞蛋白、组蛋白和cfDNA的共定位检测出NET。因此，通过脱硫素组织的免疫荧光对固定组织中NET的识别和定量优于传统的血氧林和欧辛（H&E）染色，使用明亮的场显微镜44，5。5利用免疫荧光显微镜进行的几项人体研究确定NETs为冠状动脉血栓、脑卒中、动脉炎和静脉血栓13、14、15、16、17的结构成分。13,14,15,16,17迄今为止，尚未描述一种检测和量化猫科动物血栓中NET的标准化方法。由于在猫科动物心性动脉血栓中识别NETs可能有助于未来在NET和血栓的转化研究，我们描述了NET识别和评估在猫的石蜡嵌入动脉血栓的技术。

<table>
	<tbody>
		<tr>
			<td>4,6-Diamidino-2-phenylin (DAPI)</td>
			<td>Life Technologies Corporation</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Streptavidin conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>Catalog # S11227</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-citrullinated histone H3 antibody</td>
			<td>Abcam</td>
			<td>Ab5103</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Cell Imaging System</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEFC4300</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS Imaging System Objective 10x</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4681</td>
			<td>NA 0.25, WD 6.9/7.45 mm</td>
		</tr>
		<tr>
			<td>EVOS Imaging System Objective 20x</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4682</td>
			<td>NA 0.40, WD 6.8 mm</td>
		</tr>
		<tr>
			<td>EVOS Imaging System Objective 40x</td>
			<td>ThermoFisher Scientific</td>
			<td>AMEP4699</td>
			<td>NA 0.75, WD 0.72 mm</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 488 antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>Catalog # A32723</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson Immuno Research Labs</td>
			<td>Catalog # NC9660079. Manufacturer Part # 005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutrophil elastase antibody</td>
			<td>Bioss Antibodies</td>
			<td>Bs-6982R-Biotin</td>
			<td>Rabbit polyclonal Antibody, Biotin conjugated</td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>Pierce</td>
			<td>Product # 28324. Lot # EJ64292</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive charged microscope slides</td>
			<td>Thomas Scientific</td>
			<td>Manufacturer No. 1354W-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit serum</td>
			<td>Life Technology</td>
			<td>Catalog # 10510</td>
			<td></td>
		</tr>
		<tr>
			<td>Target Retrieval Solution</td>
			<td>Agilent Dako</td>
			<td>S2367</td>
			<td>TRIS/EDTA, pH 9 (10x)</td>
		</tr>
		<tr>
			<td>TrueVIEW Autofluorescence Quenching Kit</td>
			<td>Vector Laboratories</td>
			<td>SP-8400</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of neutrophil extracellular traps in paraffin-embedded feline arterial thrombi using immunofluorescence microscopy

中性粒细胞外陷阱（NETs），由无细胞DNA（cfDNA）和蛋白质（如组蛋白和嗜中性粒细胞酶酶（NE）组成，由嗜中性粒细胞释放，以响应全身炎症或病原体。虽然NETs以前曾被证明能增强血栓形成和抑制人类和狗的纤维化，但NET在有心性动脉血栓栓塞（CATE）的猫中的作用，这是一种威胁生命的并发症，仅次于肥大心肌病，未知。一种标准化的方法识别和量化猫有氧动脉血栓中的NET，将增进我们对猫在CAT中病理作用的理解。在这里，我们描述了一种技术，用于识别主动脉分叉内甲醛固定和石蜡嵌入血栓中的NET，在尸检期间提取。与二甲苯脱硫后，主动脉部分进行了间接热诱导抗原检索。然后，通过无细胞DNA（cfDNA）、西特鲁普林语H3（citH3）和中性粒细胞酶（NE）共同定位，利用免疫荧光显微镜，发现部分阻塞、渗透和外游性NETs。为了优化血栓中NET的免疫检测，在显微镜之前使用自动荧光淬火过程限制了组织元素的自荧光。这项技术可能是研究其他物种的NET和血栓形成的有用工具，并为这种复杂情况的病理生理学提供了新的见解。

利用该协议进行除石蜡化、热诱导抗原检索和石蜡嵌血栓的双重免疫标签，我们首次确定了猫科动物CATE中的NET。主动脉分叉内的血栓通过荧光显微镜和亮场显微镜使用标准H&E染色和相位对比显微镜定位。在明亮的田间显微镜下，猫动脉血栓由红血球、白细胞、纤维蛋白和血小板组成（图3A）。虽然H&E不能染色特定的NET组件，但NET经常作为围绕附近红细胞和白细胞的各种长度?...

Watch this Scientific Journal Video about Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy at JoVE.com

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

我们描述了一种使用热诱导抗原检索和双免疫标记协议识别甲醛固定和石蜡嵌入的猫科动物心性动脉血栓中嗜中性粒细胞外陷阱（NETs）的方法。

使用免疫荧光显微镜鉴定帕芬-嵌入式猫动脉血栓中中性粒细胞外陷阱

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

这种抛物化协议，以及石蜡嵌入大动脉部分的抗原检索，是研究中性粒细胞三细胞陷阱在猫血栓形成中的作用的有用工具。通过固定和石蜡嵌入组织中的免疫荧光检测嗜中性粒细胞三细胞陷阱，优于常规组织支架（如 HNE），因为它允许同时检测更安全的DNA和细胞外蛋白。该协议可用于其他类型的血栓和其他兽医物种。在猫动脉血栓中识别嗜中性粒细胞外陷阱表明，它们可能在猫的血栓形成中发挥作用。此处介绍的议定书是准则。我们强烈鼓励调查人员调整抗原检索过程的持续时间和条件，以产生令人满意的信号。要使用自动化系统对玻璃幻灯片上的部分进行脱甲和再水化，请将玻璃滑梯放在机架中。在 100% 盐水中完全淹没三分钟。重复此步骤两次。请勿在步骤之间冲洗。在室温下完全淹没在降低乙醇浓度中，每次三次，每次三分钟。完全在去水中潜水两分钟，再重复一次。使用 TBST 处理后，将加热至 100 摄氏度的去化水填充储液罐。让蒸笼平衡 20 分钟。在热板上，以 pH 9.0 至 95 至 97 摄氏度加热含有 Tris 和 EDTA 的市售抗原检索溶液。确保不煮沸。将滑轨完全在加热抗原回收溶液中，通过蒸笼继续应用外部加热20分钟。接下来，用 TBST 将幻灯片洗三次五分钟。现在，将节放在阻塞缓冲区 1 中。在 30 至 50 rpm 的温和摇动下，在室温下孵育两小时。无需洗涤，立即将100微升稀释的兔子多克隆抗人类柠檬酸组蛋白H3抗体直接涂抹在幻灯片上。在每个部分上放置盖滑，以便抗体混合物均匀分布。在4摄氏度下孵育12至16小时，轻轻摇动。之后，用 TBST 清洗幻灯片三次，每次五分钟。要应用抗体，请使用移液器，均匀地分配与 Alexa Fluor 488 结合的 100 微升山羊抗兔抗体。用锡箔盖住幻灯片，在温和摇动下在室温下孵育幻灯片一小时。根据手稿用 TBST 清洗后，在温和摇动下在 4 摄氏度下孵育阻塞缓冲液 2 中的部分过夜。防止光线。每次用 TBST 洗三次，每次五分钟。在温和摇动下，在室温下将阻塞缓冲器 3 中的截面阻塞两个小时。如前文所述，用生物素提包龙兔抗人类嗜中性粒细胞弹性酶抗体孵育部分，在4摄氏度内孵育12至16小时。用 TBST 洗涤后，用 Alexa Fluor 594 Streptavidin 结合液孵化幻灯片，在室温下孵育一小时。之后，用TBST洗涤一次五分钟。按照制造商的指示，将100微升的自动荧光淬火溶液混合物直接涂抹到各部分上一分钟。立即用 TBST 清洗幻灯片六次，每次 10 分钟。用 100 微升 300 纳米摩尔 DAPI 覆盖每张幻灯片，在黑暗中 5 分钟。然后用TBST洗五次，每次三分钟。将一滴防淡安装介质直接涂抹在部分周围的玻璃滑梯上。将盖滑轻轻放在截面上，不产生任何气泡。让样品在黑暗中在摄氏四度下过夜。要定位血栓，请沿着主动脉、主动脉分叉和每个女性动脉的长度进行颅骨扫描，使用相位对比显微镜，目标为 10 倍。首先检查使用DAPI通道的无细胞DNA部分，其激发速度为357和44纳米。分别在德克萨斯州红色和绿色荧光蛋白通道上识别细胞外嗜中性粒细胞弹性酶和细胞外组蛋白H3。在整个图像采集过程中保持每个通道的一致曝光时间和增益，以避免像素强度饱和。中性粒细胞外陷阱是基于中性粒细胞弹性酶、细胞化组蛋白H3和无细胞DNA的共同定位。使用血氧素和 eosin 染色， 和明亮的场显微镜， 猫动脉血栓由红血球， 白细胞， 纤维蛋白和血小板组成.内特以各种长度的深紫色线网络出现，这些线围绕着附近的红细胞和白细胞。利用该协议，免疫荧光显微镜揭示了由无细胞DNA、细胞外细胞细胞基酮H3和嗜中性粒细胞弹性酶组成的大量NET。在免疫显微镜的相位对比中，血栓被定性为血管空间内的一个构造良好的结构。但是，在任何控制样本中未检测到血栓。此图显示了在 488 纳米波长上成像红细胞等凝块元素的深刻自流性。免疫标签后短暂的自荧光淬火显著提高了蛋白质共定位和 NET 检测的灵敏度，即使在红细胞丰富的区域。固定持续时间影响某些抗原的免疫反应。我们建议固定不超过24小时，以标准脱水和石蜡嵌入。或者，不含甲醇的对甲醛可用于通过甲醛氧化的甲酸和酮来限制伪影。为了防止使用来自同一物种的初级抗体时受到干扰，我们包括额外的阻断步骤，使二级抗体上剩余的结合位点饱和。研究者必须包括阴性对比，包括同型抗体、排除原抗体和免疫标记步骤以及无血栓的猫的生物对等。血栓组织极限的自动流动可以锤击中性粒细胞外陷阱的微观识别。研究者可以使用远红色的荧光团来最大限度地减少荧光。该技术可成为研究其他兽医物种中嗜中性粒细胞外陷阱的宝贵工具。这可以提供更好的理解血栓的病理生理学。

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

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

科研

免疫与感染

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

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

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

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

方法来研究中性粒细胞脂质的改变和中性粒细胞胞外陷阱随后形成

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

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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

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

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

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

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

微生物和化学刺激诱导的中性粒细胞胞外陷阱的形态学和组成分析

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

使用改良的夹心 ELISA 定量来自中性粒细胞胞外陷阱的髓过氧化物酶 DNA 和中性粒细胞弹性蛋白酶 DNA 复合物

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues (Video) | JoVE

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

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