Energy dispersive spectroscopy-scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non-opsonized Tannerella forsythia
Energy dispersive spectroscopy-scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non-opsonized Tannerella forsythia
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DOI：
10.1002/jemt.22819
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发表时间：
2017-06-01
影响因子：
2.5
通讯作者：
Honda，Masaki
中科院分区：
文献类型：
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作者：
Moriguchi，Keiichi;Hasegawa，Yoshiaki;Honda，Masaki
我们研究了人多形核白细胞(PMN)与显示抗血清表层(S层)的非调理福岛坦纳氏菌ATCC 43037之间的关系。当中性粒细胞与T混合时。连翘苷悬浮液，细胞吞噬T。连翘叶。用光学显微镜观察到硝基蓝四氮唑(NBT)的还原反应，用电子显微镜观察到指示过氧化氢生成的过氢氧化铈(Ce)的沉积。我们研究了S层的高分子量蛋白质与Ce反应(Fort.用电子显微镜免疫标记)。免疫金颗粒定位于中性粒细胞内和细胞表面，标记在与S层相同的Ce反应部位。用能谱仪-扫描电子显微镜对吞噬细胞进行Ce和N(S层免疫细胞化学)元素分析。也就是说，N的元素图谱和EDS分析似乎反映了3，3‘-二氨基联胺-四盐酸盐(DAB)与辣根过氧化物酶(HRP)标记的二抗反应所检测到的相同部分的存在，而不是免疫金标记法。我们专注于使用EDS-STEM来可视化DAB反应产生的N的存在。在一组平行的实验中，我们使用能谱扫描电子显微镜对相同的吞噬细胞进行了Ce和金(来自S层的免疫金标记物)元素分析。
We investigated the association between human polymorphonuclear leukocytes (PMNs) and non‐opsonizedTannerella forsythiaATCC 43037 displaying a serum‐resistant surface layer (S‐layer). When PMNs were mixed withT. forsythiain suspension, the cells phagocytosedT. forsythiacells. Nitro blue tetrazolium (NBT) reduction, indicative of production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H2O2production, was observed by electron microscopy. We examined the relationship between high‐molecular‐weight proteins of the S‐layer and Ce reaction (forT. forsythiaphagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce‐reacted sites where the S‐layer was present. We then used energy dispersive spectroscopy (EDS)‐scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S‐layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)‐conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS‐STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS‐STEM to perform Ce and gold (Au; from immunogold labeling of the S‐layer) elemental analysis on the same phagocytosing cells.