Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy

Abstract

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. T. forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3’diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.