Radermecker Coraline, Hego Alexandre, Delvenne Philippe, Marichal Thomas
Laboratory of Immunophysiology, Grappe Interdisciplinaire de Genoproteomique Appliquee (GIGA) Institute, Liege University, Liege, Belgium.
Faculty of Veterinary Medicine, Liege University, Liege, Belgium.
Bio Protoc. 2021 Sep 20;11(18):e4159. doi: 10.21769/BioProtoc.4159.
Neutrophils are one of the first innate immune cells recruited to tissues during inflammation. An important function of neutrophils relies on their ability to release extracellular structures, known as Neutrophil Extracellular Traps or NETs, into their environment. Detecting such NETs in humans has often proven challenging for both biological fluids and tissues; however, this can be achieved by quantitating NET components (, DNA or granule/histone proteins) or by directly visualizing them by microscopy, respectively. Direct visualization by confocal microscopy is preferably performed on formalin-fixed paraffin-embedded (FFPE) tissue sections stained with a fluorescent DNA dye and antibodies directed against myeloperoxidase (MPO) and citrullinated histone 3 (Cit-H3), two components of NETs, following paraffin removal, antigen retrieval, and permeabilization. NETs are defined as extracellular structures that stain double-positive for MPO and Cit-H3. Here, we propose a novel software-based objective method for NET volume quantitation in tissue sections based on the measurement of the volume of structures exhibiting co-localization of Cit-H3 and MPO outside the cell. Such a technique not only allows the unambiguous identification of NETs in tissue sections but also their quantitation and relationship with surrounding tissues. Graphic abstract: Graphical representation of the methodology used to stain and quantitate NETs in human lung tissue.
中性粒细胞是炎症期间最早募集到组织中的固有免疫细胞之一。中性粒细胞的一项重要功能依赖于它们向周围环境释放细胞外结构的能力，这种结构称为中性粒细胞胞外诱捕网（NETs）。在人体中，检测生物体液和组织中的此类NETs往往具有挑战性；然而，这可以分别通过定量NET成分（如DNA或颗粒/组蛋白）或通过显微镜直接观察来实现。通过共聚焦显微镜直接观察，最好在经石蜡去除、抗原修复和通透处理后，用荧光DNA染料以及针对NETs的两种成分髓过氧化物酶（MPO）和瓜氨酸化组蛋白3（Cit-H3）的抗体进行染色的福尔马林固定石蜡包埋（FFPE）组织切片上进行。NETs被定义为对MPO和Cit-H3呈双阳性染色的细胞外结构。在此，我们提出一种基于软件的新型客观方法，用于在组织切片中对NET体积进行定量，该方法基于测量细胞外呈现Cit-H3和MPO共定位的结构的体积。这种技术不仅能明确识别组织切片中的NETs，还能对其进行定量以及确定它们与周围组织的关系。图形摘要：用于对人肺组织中的NETs进行染色和定量的方法的图形表示。
