Visualization of neutrophil extracellular traps in TEM

Neutrophil extracellular traps (NETs) have recently been described as an important innate defence mechanism in inflammation. However, routine electron microscopic staining techniques faintly stain NETs and are therefore insufficient for enabling a distinction between these and the host cell debris as well as proteins regularly present at the site of inflammation. In order to test suitable electron microscopic staining techniques, NETs induced ex vivo via phorbol myristate were absorbed on formvar. Four types of drop-on-grid positive staining were used: osmium tetroxide (Os), osmium tetroxide-uranyl acetate-lead citrate (Os-U-Pb), ruthenium red-osmium tetroxide (RR-OsO4), and cuprolinic blue enhanced by sodium tungstate (CB-WO4). We observed no staining of NETs using Os, faint staining with Os-U-Pb, better but still weak staining with CB-WO4 and outstanding staining with RR-OsO4. Furthermore, RR-OsO4 staining also enables the observation of bacterial fimbriae-mediated adhesion, which is possibly responsible for the ability of NETs to bind bacteria. Thus, the offered RR-OsO4 staining technique may facilitate the study of the NETs-bacterial interactions.