Epon resin

Biopsy specimens were immersed in ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) immediately after their extraction and sectioning, and fixed for 4 h at 4 °C, carefully handling samples to avoid ex vivo artefacts. After washing in cacodylate buffer, kidney fragments were then postfixed in 1% osmium tetroxide for 1 h, dehydrated through ascending grades of alcohol, and embedded in Epon resin (Electron Microscopy Science, Hatfield, PA). Ultrathin sections (60 to 100 nm) were cut on an EM UC7 ultramicrotome (Leica Microsystems, Mannheim, Germany), stained with uranyl acetate and lead citrate, and examined with TEM (Morgagni 268D, Philips, Brno, Czech Republic).

Dissected TA muscles were fixed in 2.5% glutaraldehyde diluted in 0.1M phosphate buffer, pH 7.4. They were further post-fixed in 2% OsO4, gradually dehydrated in acetone including a 2% uranyl staining step in 70% acetone, and finally embedded in Epon resin (Electron Microscopy Sciences). After uranyl and lead citrate staining, ultrathin sections were examined with a Philips CM120 electron microscope and images were acquired using a SIS Morada digital camera.

Parasites were fixed at room temperature with 2% formaldehyde/2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). The fixed parasites were then decanted overnight on ACLAR (Electron Microscopy Sciences) film precoated with 0.01% poly-L-lysine. The ACLAR films were washed three times with 0.1 M sodium cacodylate (pH 7.2) and then fixed with 1% osmium tetroxide in the same buffer at room temperature. The preparation was then washed with cacodylate buffer followed by three washes with water. Subsequently, the ACLARfilms were treated with 0.4% uranyl acetate, gradually dehydrated in a series of ethanol solutions, and embedded in Epon resin (Electron Microscopy Sciences). After sectioning to produce consecutive sections, the material was stained with uranyl acetate and lead citrate, mounted on Formvar grids, and observed in a JEOL 1200 EX II transmission electron microscope at 80 kV.

The OMVs isolated from cultures grown with and without ampicillin, amikacin, chloramphenicol, colistin, meropenem, minocycline, imipenem, polymyxin B, and tigecycline were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in PBS and thoroughly mixed for TEM analysis. Fixed samples were washed in 0.1 M sodium cacodylate (pH 7.24), postfixed with buffered 2% OsO₄, washed with water, and dehydrated in a graded ethanol series (25 to 100%) with 25% increments. The samples were filtered in a graded series of Epon resin (Ted Pella Inc., Redding, CA, USA) for 2 days, embedded in fresh Epon resin, and polymerized at 60°C for 48 h. Purified OMV pellets were resuspended in 2% paraformaldehyde and allowed to free-float onto a 200-mesh carbon-coated Formvar nickel grid (Electron Microscopy Sciences, Hsin An Instruments Co. Ltd., Taiwan) for 5 min. The excess solution was dried off with filter paper, and the sample grid was then floated on a 10-μL droplet of 1% aqueous uranyl acetate for 30 s. The stain was removed with filter paper, and the sample was air dried. Finally, the samples were imaged using a Hitachi (Tokyo, Japan) HT7700 transmission electron microscope.

Light and electron microscopes were used to assess cell morphology, following the procedures modified from our previous study [28 (link)].
Cell morphology was observed under a light microscope (Olympus, Tokyo, Japan) at 1000× magnification. The cells were stained by Liu’s staining method; using Liu A solution for 45 s, followed by Liu B solution for 90 s. Cells were collected, washed, and fixed with 2.5% glutaraldehyde in cacodylate buffer for 30 min for TEM. Cells were fixed with osmium tetroxide (1%) and embedded in Epon resin (Electron Microscopy Science, Hatfield, PA, USA). Samples were stained with 0.5% toluidine blue and examined under a light microscope. Semi-thin and ultra-thin sections were cut. Ultra-thin sections were stained with 2% uranyl acetate and Reynold’s lead citrate and visualized using TEM with a digital camera (JEM-1200EXII, JEOL Co., Tokyo, Japan).