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.
Immunogold Labeling of GMMA Vesicles
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.
Corresponding Organization : Toscana Life Sciences
Other organizations : Boston VA Research Institute, Harvard University, Luigi Sacco Hospital, University of Milan, University of Siena, Agency for Science, Technology and Research
Variable analysis
- GMMA concentration (100 µg/mL)
- GMMA diameter (measured manually in comparison with the scale bar)
- Immunogold labeling of fHbp and O-antigen (OAg)
- Formvar/carbon-coated grids
- Uranyl acetate for negative staining
- Anti-fHbp polyclonal serum (diluted 1:400)
- Anti-OAg monoclonal antibody (diluted 1:1000)
- Gold-labeled anti-mouse secondary antibody (diluted 1:40)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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