Summary

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Published: August 18, 2023
doi:

Summary

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。

Abstract

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。

Introduction

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6,7,8NETs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 3,13,16,17,18,19,20,21,22,23,24,2526,27.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。

Protocol

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?…

Representative Results

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?…

Discussion

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?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

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。

Materials

         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

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Schoenfeld, L., Appl, B., Pagerols-Raluy, L., Heuer, A., Reinshagen, K., Boettcher, M. Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues. J. Vis. Exp. (198), e65272, doi:10.3791/65272 (2023).

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