Immunogold Labeling of Outer Membrane Vesicles

Electron microscopy of ultrathin cryosections of LB agar cultures of strains 5791/99 and 493/89, or of ultrathin cryosections of OptiPrep-purified 5791/99 OMVs was performed as described [10 (link)]. Briefly, overnight LB agar cultures harvested into PBS or a pool of OptipPrep fractions 1 to 8 of 5791/99 OMVs were fixed with 2% paraformaldehyde and 0.2% glutaraldehyde and processed using the method of Tokuyasu [85 (link)]. Ultrathin (50 nm) frozen sections were cut and immunogold-stained with anti-E. coli O157 LPS antibody (culture sections), or with anti-Stx2a, anti-CdtV-A, anti-CdtV-B, anti-EHEC-Hly, and anti-H7 antibody (OMV sections) and Protein A Gold (15 nm) (Department of Cell Biology, University Medical Center, Utrecht, The Netherlands). Staining with Protein A Gold alone, without first antibodies, served as a control of specificity of the immunogold staining. For negative staining, OMVs in OptiPrep fractions of 5791/99 and 493/89 OMVs were fixed and stained with 0.5% uranyl acetate. Samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, The Netherlands) and photographed (Ditabis imaging plates).