Immunogold Labeling of Francisella in BMDMs
Corresponding Organization : University of Defence
Other organizations : Czech Academy of Sciences, Institute of Molecular Genetics, Czech Academy of Sciences, Charles University
Variable analysis
- Duration of infection (2 h, 12 h, 24 h)
- Localization and density of immunolabeling of GapA (FTT1368) protein in bacterial cells and host cells
- BMDMs seeded on glass coverslips at density 1 × 10^6 cells per well in 24-well tissue culture
- Infection with F. tularensis FSC200 as described
- Washing in pre-warmed Sörensen's buffer
- Fixation in 2.5% paraformaldehyde with 0.25% glutaraldehyde in Sörensen's buffer for 1 h at room temperature
- Quenching of free aldehyde groups with 0.02 M glycine in Sörensen's buffer for 10 min
- Dehydration in a series of ethanol and embedding into LR White resin
- Polymerization for 72 h under UV light at 4 °C
- Preparation of 80 nm ultrathin sections using Ultramicrotome Leica EM UC6 with a diamond knife
- Mounting of sections on formvar-coated 3.05 mm gilded copper slots
- Immunogold-labeling following a conventional protocol
- Examination in FEI Morgagni 268 TEM or Jeol JEM-1400 FLASH TEM operated at 80 kV
- Use of primary rabbit anti-FTT1368 (GapA) antibody at dilutions 1:25 and 1:100 and secondary goat anti-rabbit IgG (H + L) antibody coupled with 12 nm colloidal gold particles
- Use of uninfected BMDMs as a control of specificity of the GapA antibody
- Usual technical negative controls with an omitted primary antibody
- Uninfected BMDMs used as a control of specificity of the GapA antibody
- Usual technical negative controls with an omitted primary antibody
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