Jem 1011

Cells treated with Ses at its MIC₈₀ value were observed by using SEM (Zeiss EVOMA10) and TEM (JEOL JEM-1011). The cells (~10⁶ cells) were administered to the media with and without Ses and were incubated for 24 h at 30°C. Sample preparation and analysis were performed by using the method as described elsewhere [16 (link), 17 (link)]. Briefly, all cells were fixed with 2% glutaraldehyde in 0.1% phosphate buffer for 1 h at room temperature (20°C), washed with 0.1 M phosphate buffer (pH 7.2), and postfixed with 1% OsO₄ in 0.1 M phosphate buffer for 1 h at 4°C. Then the cells were dehydrated in acetone, dropped on round glass coverslip with hexamethyldisilazane (HMDS), dried at room temperature, and then sputter coated with gold and observed under the SEM (Zeiss EVOMA10) at 30 K magnificationand TEM (JEOL JEM-1011) at 10 K magnification.

For TEM, the animals were fixed in 2.5% glutaraldehyde in phosphate buffer saline and were postfixed for 2 h in 1% osmium tetroxide in the same buffer saline. After the specimens were washed with the same buffer saline, they were transferred through an ethanol series and stored in 70% ethanol at 4 °C. Further preparation included dehydration in an ethanol series and acetone, and embedding in Spurr resin (Sigma). Semithin and thin sections were cut with a Leica UC-6 ultratome (Leica, Germany), then were stained with methylene blue, observed with a Zeiss Axioplan2 microscope, and photographed with an AxioCam HRm camera. Ultrathin sections were stained in uranyl acetate followed by lead nitrate and were examined with JEM-1011 JEOL and JEM-100 B-1 JEOL transmission electron microscopes (JEOL, Akishima, Japan).

For transmission electron microscopy (TEM), the animals were fixed overnight at 4 °C in 2.5 % glutaraldehyde in phosphate buffer saline with addition of NaCl (pH 7.4, osmolarity 830 milliosmols) [44 ] and were postfixed for 2 h in 1 % osmium tetroxide in the same buffer saline. After the specimens were washed with the same buffer saline, they were transferred through an ethanol series and stored in 70 % ethanol at 4 °C. Further preparation included dehydration in an ethanol series and acetone, and embedding in Epon-Araldite resin (Electron Microscopy Sciences, Fort Washington, PA, USA). Semithin and thin sections were cut with a Ultracut-R Leica ultratome (Leica, Germany). Semithin sections were stained with methylene blue, observed with a Zeiss Axioplan2 microscope, and photographed with an AxioCam HRm camera. Ultrathin sections were stained in uranyl acetate followed by lead nitrate and were examined with JEM-1011 JEOL and JEM-100 B-1 JEOL transmission electron microscopes (JEOL, Akishima, Japan).
Three-dimensional reconstructions were generated using Amira version 5.2.2 software (Bitplane, Zurich, Switzerland). TEM micrographs and Z-projections were processed in Adobe Photoshop (CS3 7.0.1., Adobe Systems) to prepare panoramas and combinations of Z-projections.

The head regions with tentacles were fixed overnight at 4 °C in a 2.5% solution of glutaraldehyde in 0.2 M phosphate buffer (PBS). The heads were then washed in 0.2 M PBS for 4 h with three changes and postfixed in 1% OsO₄ in 0.2 M PBS for 3 h at room temperature (RT) with gentle rotation. The specimens were then dehydrated in an increasing series of ethanol concentrations (from 15 to 96%) and isopropanol. They were subsequently infiltrated in a mixture of isopropanol and Spurr resin for 3 days and then embedded in pure Spurr resin at 60 °C for 24 h.
The anterior part of the body of two adults embedded in resin were used to prepare a complete series of 1-μm (semi-thin) and 70-nm (thin) resin sections with a Leica UC 7 ultramicrotome (Leica Microsystems, Wetzlar, Germany). The semi-thin sections were stained with methylene blue and examined with a Zeiss Axioplan2 light microscope equipped with an AxioCam HRm camera (Carl Zeiss Microscopy, LLC, USA). Semi-thin sections were used for description of gross anatomy and for 3D reconstructions. The thin sections were stained with uranyl acetate and lead citrate and were examined with a JEM-1011 JEOL or a JEM-100 B-1 JEOL transmission electron microscope (JEOL, Akishima, Japan).

The exosomes were extracted from patient sera, using the sequence of centrifugation and ultracentrifugation described in a previous setting protocol [40 (link)]. An aliquot was used immediately for exosome characterization; then, the other part was stored at −80 °C until the protein analysis was carried out. The characterization of exosomes was performed using the Jeol Jem-1011 (JEOL USA, Inc, Peabody, MA, USA), a transmission electron microscope working at an accelerating voltage of 100 kV, and the Olympus Quemesa Camera (11 Mpx) (Olympus, Shinjuku-ku, Tokyo 163-0914, Japan) to acquire proper images. The staining of samples was performed according to the experimental procedure reported in a previous paper [41 (link),42 (link)]. The hydrodynamic diameter, ζ-potential, and stability with the corresponding polydispersity index (PDI) were investigated with the Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK), a dynamic light scattering (DLS) system. The operative instrument condition was a 4 mW He-Ne laser as a light source (wavelength λ = 633 nm). A disposable folded capillary cell, DTS1070 (Malvern Instruments Ltd., Worcestershire, UK) was used, as already reported by Depalo et al. [43 (link)]. Three consecutive measurements were performed on each sample to obtain data, reported as average value ± standard deviation.

¹H NMR spectra were obtained using a Varian 500-MHz spectrometer with dimethyl sulfoxide (DMSO)-d6 as solvent and tetramethylsilane (TMS) as the internal standard. The Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 10 (Perkin Elmer, Waltham, MA, USA). FTIR spectra were collected in solid state and taken from 400 to 4000 cm⁻¹ with a resolution of 4 cm⁻¹ for 64 scans. The surface morphology and size characterizations were performed using transmission electron microscopy (TEM, JEOL JEM-1011 (JEOL Inc, Peabody, MA, USA)) and scanning electron microscope (SEM, TESCAN LYRA3) (TESCAN, Warrendale, PA, USA). The sample was prepared by dropping the solution of nanoparticles onto carbon-coated copper grids for TEM observation. SEM samples were prepared by dropping the solution of nanoparticles onto carbon tape. The size analysis from TEM and SEM images was performed using ImageJ software (version 1.52a). Energy-dispersive X-ray spectroscopy (EDS) elemental mapping and line-profile elemental analysis were performed using a Noran system 7 by thermo scientific operated at 20 KV. UV-vis spectra were measured with a UV-2600 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The iron concentration was analyzed by using a Varian 820 Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (Varian, Mulgrave VIC, Australia).