title: Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.
abstract: 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.
authors.affiliation: Department of Oral Anatomy, Division of Oral Infections and Health Sciences, Asahi University School ofDentistry, 1851 Hozumi, Mizuho, Gifu 501 - 0296 Japan. keiichi@dpc.aichi-gakuin.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Moriguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Mitamura
authors.forename: J
authors.initials: J
authors.lastname: Iwami
authors.forename: Y
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: N
authors.initials: N
authors.lastname: Higuchi
authors.forename: Y
authors.initials: Y
authors.lastname: Murakami
authors.forename: H
authors.initials: H
authors.lastname: Maeda
authors.forename: F
authors.initials: F
authors.lastname: Yoshimura
authors.forename: H
authors.initials: H
authors.lastname: Nakamura
authors.forename: N
authors.initials: N
authors.lastname: Ohno
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis; D001439:Bacteroides / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure*; D002949:Citraconic Anhydrides / Q000737:chemistry*; D006361:Heating / Q000295:instrumentation*; D006735:Horseradish Peroxidase / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods*; D008562:Membrane Glycoproteins / Q000032:analysis*; D046032:Microscopy, Energy-Filtering Transmission Electron / Q000379:methods*; D008872:Microwaves
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002949:Citraconic Anhydrides; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D015100:3,3'-Diaminobenzidine; D006735:Horseradish Peroxidase