The GMMA were adsorbed to Formvar/carbon-coated grids, negatively stained with uranyl acetate, as described previously [48 (link)], and subsequently observed with a Tecnai G2 Spirit transmission electron microscope (FEI, Eindhoven, The Netherlands) operating at 80 kV. Electron micrographs were recorded at a nominal magnification of X87,000. GMMA diameters were manually measured in comparison with the scale bar. Immunogold labeling was obtained by staining with anti-fHbp or anti-OAg mice polyclonal sera.
For analysis by immunogold staining, a 5 µL aliquot of GMMA with a final concentration of 100 µg/mL were adsorbed to 300-mesh nickel grids, blocked in PBS with 0.5% bovine serum albumin (BSA) and incubated with primary anti-fHbp polyclonal serum (diluted 1: 400 in PBS with 1% BSA) or primary anti-OAg mAb (AbCAM, diluted 1:1000) for 1 h. Grids were washed several times in PBS with 1% bovine serum albumin and incubated with gold-labeled anti-mouse secondary antibody (diluted 1: 40 in PBS with 1% bovine serum albumin) for 1 h. After several washes with distilled water the grids were negatively stained and analyzed using a TEM FEI Tecnai G2 spirit microscope.
For analysis by immunogold staining, a 5 µL aliquot of GMMA with a final concentration of 100 µg/mL were adsorbed to 300-mesh nickel grids, blocked in PBS with 0.5% bovine serum albumin (BSA) and incubated with primary anti-fHbp polyclonal serum (diluted 1: 400 in PBS with 1% BSA) or primary anti-OAg mAb (AbCAM, diluted 1:1000) for 1 h. Grids were washed several times in PBS with 1% bovine serum albumin and incubated with gold-labeled anti-mouse secondary antibody (diluted 1: 40 in PBS with 1% bovine serum albumin) for 1 h. After several washes with distilled water the grids were negatively stained and analyzed using a TEM FEI Tecnai G2 spirit microscope.