Immunogold Labeling of Francisella in BMDMs

BMDMs seeded on glass coverslips at density 1 × 10⁶ cells per well in 24-well tissue culture were infected with F. tularensis FSC200, as described above, for 2, 12, and 24 h. Following infection, the cells were briefly washed in pre-warmed Sörensen’s buffer (0.1 M Na/K phosphate buffer, pH 7.2–7.4) and fixed in 2.5% paraformaldehyde with 0.25% glutaraldehyde (Thermo Fisher Scientific) in Sörensen’s buffer for 1 h at room temperature (RT). All subsequent steps were performed on ice. After several washes, free aldehyde groups were quenched with 0.02 M glycine in Sörensen’s buffer for 10 min. Samples were then dehydrated in a series of ethanol (Lach-Ner, Neratovice, Czech Republic) and embedded into LR White resin (Sigma-Aldrich). After polymerization for 72 h under UV light at 4 °C, 80 nm ultrathin sections were prepared using the Ultramicrotome Leica EM UC6 (Leica Microsystems, Wetzlar, Germany) equipped with a diamond knife (Diatome, Biel, Switzerland). The sections were mounted on formvar-coated 3.05 mm gilded copper slots (Agar scientific, Essex, UK), immunogold-labeled following a conventional protocol [29 (link)], and examined in an FEI Morgagni 268 transmission electron microscope (TEM) with Mega View III CCD camera (Olympus Soft Imaging Solutions, Münster, Germany) or in a Jeol JEM-1400 FLASH TEM equipped with 2kx2k Matataki CMOS camera. Both TEMs were operated at 80 kV. Antibodies used for immunolabeling were primary rabbit anti-FTT1368 (GapA) antibody at dilutions 1:25 and 1:100 (Apronex, Vestec, Czech Republic) and secondary goat anti-rabbit IgG (H + L) antibody coupled with 12 nm colloidal gold particles (Jackson ImmunoResearch Laboratories Inc., Baltimore Pike, West Grove, PA, USA; 111-205-144; dilution 1:40). Uninfected BMDMs were used as a control of specificity of the GapA antibody, and usual technical negative controls were performed with an omitted primary antibody. The density of immunolabeling in specified compartments of bacterial cells and host cells was calculated as a ratio between the number of gold nanoparticles in a specified region and its area in µm². The areas were measured in ELLIPSE Software, version 2.0.8.1 (ViDiTo, Kosice, Slovakia). The calculation of statistical significance was based on a comparison of labeling density values and variance in individual cells between analyzed variants.