Tecnai g2 spirit

Strains were grown as previously described. Approximately 2 × 10⁹ bacterial cells were resuspended in 1 mL of PBS and fixed with 4% paraformaldehyde. 5 µL of each sample were applied to a 200-square mesh nickel grid coated with a thin carbon film. Samples were blocked with PBS + 1% BSA and then incubated for 1 h at RT with the primary antibody (diluted 1:250 in the blocking solution). Grids were washed twice and incubated with gold labelled anti-mouse secondary antibodies (diluted 1:40 in 1% PBS-BSA) for 1 h. Samples were washed in distilled water and observed using TEM FEI Tecnai G2 Spirit operating at 100 kV and equipped with a CCD Olympus SIS Morada camera (Olympus, Shinjuku, Tokyo, Japan). Images were acquired and processed using the iTemm (OSIS, Olympus, Shinjuku, Tokyo, Japan) software.
To verify the presence of aggregates in T7expressIq (pET15b)INP-NmBamE, post-embedding experiments were performed. The sample was divided into aliquots and fixed O/N at 4 °C in 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2.5% formaldehyde and then post-fixed in 1% OsO₄. Samples were then dried by the critical point method using CO₂ in a Balzers Union CPD 020. The dried samples were embedded in LRWhite resin and stained with uranyl acetate and lead citrate.

Purified exosomes were fixed with 2% paraformaldehyde. A 20 μl drop of the suspension was loaded onto a formvar coated grid, negatively stained with 2% aqueous uranyl acetate for 2 minutes, and examined under a transmission electron microscope FEI Tecnai G2 Spirit using a digital camera Morada (Olympus Soft Image Solutions).