人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Lavinia Schoenfeld; Birgit Appl; Laia Pagerols-Raluy; Annika Heuer; Konrad Reinshagen; Michael Boettcher

doi:10.3791/65272

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Abstract

Immunology and Infection

人和小鼠组织中性粒细胞胞外陷阱的免疫荧光成像

Published: August 18th, 2023

DOI:

10.3791/65272

Lavinia Schoenfeld¹, Birgit Appl¹, Laia Pagerols-Raluy¹, Annika Heuer³, Konrad Reinshagen¹, Michael Boettcher^1,2

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)

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

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。

Erratum

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

An erratum was issued for: Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues. The Authors section was updated from:

Lavinia Schöenfeld¹
Birgit Appl¹
Laia Pagerols-Raluy¹
Annika Heuer³
Konrad Reinshagen¹
Michael Boettcher^1,2
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 Boettcher^1,2
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)

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