Leo 906e

Infected monolayers and ileum fragments were first fixed in 2% glutaraldehyde (EMS, USA) for at least 24 h at 4°C. After primary fixation, cells and fragments were washed 3 times with PBS (10 min) and subjected to secondary fixation with 1% osmium tetroxide (EMS, USA) in 0.1 M sodium cacodylate buffer for 30 min. After being washed three times with distilled water, preparations were dehydrated through a graded ethanol series (50%, 75%, 85%, 95% and 100%), and propylene oxide (100%). Preparations were then gradually embedded in Araldite, which was allowed to polymerize for 24–48 h at 60°C. Ultrathin sections were placed on Formvar (EMS, USA) coated 200 mesh copper grids and stained with 4% aqueous uranyl acetate (Merck, Germany) and Reynold's lead citrate (Merck, Germany). Grids were examined under TEM (LEO 906E– Zeiss, Germany) at 80 kV [48 (link)].

For TEM images of NGB–NPs, the samples were prepared with negative staining. The dispersion was incubated in a grid with a carbon support film for 5 min in a Petri dish and contrasted with 1% uranyl acetate in aqueous solution. Then, these samples were mounted and visualized on a TEM microscope LEO 906E (Carl Zeiss LEO 906E, Jena, Germany).
For SEM images, a drop of the colloidal dispersion of NGB–NPs was deposited on the corresponding support for the FESEM (pin stub mount, Hitachi Ltd., Chiyoda, Tokyo, Japan), allowing it to dry at room temperature. Subsequently, it was sputtered with carbon by means of a carbon coater (Polaron CC7650, Quorum Technologies Ltd., Laughton, East Sussex, UK) and visualized on a SEM microscope (Hitachi S-510, Hitachi Ltd., Chiyoda, Tokyo, Japan).
Both microscopes are located at the Scientific Instrumentation Center of the University of Granada (Granada Spain).

After growing overnight in DMEM at 37°C, bacterial cultures were centrifuged, washed with PBS and fixed with 4% formaldehyde. After fixation, preparations were washed, blocked with 0.2% BSA in PBS, and incubated overnight with rabbit anti-EspA antibody (1:10 dilution in PBS) at 4°C. Preparations were subsequently washed with PBS and incubated with goat anti-rabbit antibody labeled with 10 nm colloidal gold particles (Sigma-Aldrich) diluted 1:10 in PBS, for 3 h at room temperature. After further washings, preparations were negatively stained (or not) with 2% uranyl acetate in water, in order to facilitate counting and measurement of the EspA filaments and placed onto Formvar-coated nickel grids. After air-dried, preparations were then analyzed under transmission electron microscopy (TEM) (LEO 906E – Zeiss, Germany) at 80 kV.

An LS instrument (Scatterscop Qudix, Seoul, South Korea) was used to determine the median particle size and size distribution of nanoparticles. Dynamic light scattering was performed at a 90° and at temperature of 25°C. All samples were analyzed in triplicate, and their average was reported. TEM was performed using the Transmission electron microscope (Zeiss LEO 906 E, Freiburgim Breisgau, Germany) at an accelerating voltage of 80 kV. For this purpose, the samples were prepared by depositing a drop of nanoparticles containing phosphotungstic acid (2%) onto copper grids, and the extra liquid was removed by a filter paper. Then, the grids were allowed to air dry at room temperature. The structure of nanoparticles was investigated using SEM (Zeiss LEO 1455 VP, Freiburg im Breisgau, Germany) at 30k V acceleration voltage.

Samples of an induced C. merolae culture were gently pelleted and resuspended in cacodylate buffer (75 mM cacodylate, pH 7.0) supplemented with 2% glutaraldehyde. Samples were fixed for 3.5 h on ice. Cells were immobilised in 2% agarose in cacodylate buffer before fixing with 1% OsO4 overnight at 4 °C. After washing with cacodylate buffer three more times, samples were dehydrated through a graded series of acetone in cacodylate buffer (30, 50, 70, 90, 100%) at 4 °C with two additional changes in the 100% at room temperature. Cells were embedded into epoxy resin (ERL-4221D, D.E.R 736, nonenyl succinic anhydride, dimethylaminoethanol) following a protocol by Spurr [36 (link)].
Ultra-thin sections (~80 nm) were prepared on a microtome equipped with a diamond knife (Ultracut E, Reichert-Jung, Heidelberg, Germany) and transferred to copper grids (150 mesh) with a film of polyvinyl butyral (Mowital). The probes were contrasted with solutions of 2% uranyl acetate and 2% lead citrate for 10 min each. Images were acquired at 100kV on a transmission electron microscope (LEO 906E, Zeiss, Jena, Germany) equipped with a CCD camera (MultiScan 794, Gatan, Pleasanton, CA, U.S.A.).

A morphological analysis was performed in the right lungs of 3 mice per group, as previously described [54 (link)]. Briefly, in at least in 3 experiments, lungs were differentially fixed for either ultrastructural or histopathology analysis by intratracheal perfusion, and prepared for examination under a transmission electron microscope (Zeiss LEO 906E) or light microscope (Axiostar Plus, Zeiss, Germany).

NSe particles (5–50 nm) were purchased from Sigma Aldrich (USA), separated by centrifugation, and dispersed in an aqueous medium by means of sonication. Meanwhile, TEM (Zeiss, LEO 906E, Germany) was used to examine the NSe particles’ shape and size [21 (link)].