We investigated the association between human polymorphonuclear leukocytes (PMNs) and non-opsonized Tannerella forsythia ATCC 43037 displaying a serum-resistant surface layer (S-layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H2O2 production, was observed by electron microscopy. We examined the relationship between high-molecular-weight proteins of the S-layer and Ce reaction (for T. forsythia phagocytosis) 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.
We investigated the association between leukocytes and T. forsythia (T.f.) by EDS-STEM. We detected Ce, H2O2 production, and nitrogen (S-layer from T. f, DAB with HRP instead of immunogold labeling) elements on the same sites of leukocyte cytoplasm.
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