Moriguchi K, Mitamura Y, Iwami J, Hasegawa Y, Higuchi N, Murakami Y, Maeda H, Yoshimura F, Nakamura H, Ohno N
Department of Oral Anatomy, Division of Oral Infections and Health Sciences, Asahi University School ofDentistry, 1851 Hozumi, Mizuho, Gifu 501 - 0296 Japan.
Biotech Histochem. 2012 Nov;87(8):485-93. doi: 10.3109/10520295.2012.687117. Epub 2012 Sep 18.
Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.
福赛坦氏菌（原名牙龈拟杆菌）是一种革兰氏阴性厌氧菌，在牙周疾病进展中起作用，具有独特的细菌蛋白谱。其特征在于有两条分子量超过200 kDa的独特蛋白带。还已知它有一个典型的表层（S层），由外膜外规则排列的亚基组成。我们使用化学固定的电子显微镜免疫标记以及包括在微波炉中加热或用柠康酸酐高压灭菌的抗原修复程序，研究了高分子量蛋白与S层之间的关系。免疫金颗粒清晰地定位在最外层细胞表面。我们还使用能量过滤透射电子显微镜（EFTEM）在用1%柠康酸酐进行微波抗原修复后观察3,3'-二氨基联苯胺四盐酸盐（DAB）反应产物。EFTEM对氮的电子光谱图像（ESI）的三窗口方法反映了与辣根过氧化物酶（HRP）偶联的二抗进行DAB反应所证明的部分的存在，而不是免疫金颗粒。净氮的映射模式局限于最外层细胞表面。
