Epon araldite mixture

After fixation and dehydration, the samples were incubated with propylene oxide prior to exposure to propylene oxide/Epikote [13 (link),14 (link)]. The blocks were embedded with Epon-Araldite mixture (Electron Microscopy Sciences, Hatfield, PA, USA) and sectioned. The sliced samples were then examined under a transmission electron microscope (HITACHI-7000, Hitachi, Japan). For immunogold labeling, the sections of the cells on nickel grids were treated with 10% H₂O₂ for 10 min and then blocked with SuperBlock^TM blocking buffer (Thermo Fisher Scientific Inc.) for 30 min followed by mouse monoclonal anti-FTH1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and rabbit polyclonal anti-NCOA4 (Abcam) antibody treatment overnight. The grids were hybridized with goat polyclonal anti-mouse (20 nm) and goat polyclonal anti-rabbit (12 nm) secondary antibodies containing gold particles (Abcam) for 1 h. The sections were then stained with saturated uranyl acetate and lead citrate. The images were examined under a transmission electron microscope.

Cells were incubated with propylene oxide and then exposed to propylene oxide/Epikote. The blocks embedded with the Epon-Araldite mixture (Electron Microscopy Sciences, Hatfield, PA, USA) were sectioned for imaging under a transmission electron microscope (HITACHI-7000, Hitachi, Tokyo, Japan). For immunogold labeling, the ultrathin sections of the cells on the nickel grids were blocked with the SuperBlock^TM blocking buffer (Thermo Fisher Scientific Inc.), followed by incubation with mouse monoclonal anti-FTH1 (1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA, sc-376594) and rabbit polyclonal anti-nuclear receptor co-activator 4 (NCOA4, 1:100, Abcam, ab222071) antibodies. The grids incubated with gold-containing goat polyclonal anti-mouse (20 nm; 1:10, Abcam, ab27242) and anti-rabbit (12 nm; 1:10, Abcam, ab105298) secondary antibodies were stained with saturated uranyl acetate and lead citrate, respectively. The images were examined under a transmission electron microscope (HITACHI-7000, Hitachi, Tokyo, Japan).

The samples of cellulite harvested during surgical treatment of cellulite, in living people, were fixed with glutaraldehyde 2% and were post-fixed in 1% osmium tetraoxide (OsO₄) aqueous solution for 2 h, dehydrated in graded concentrations of acetone and immersed in an Epon–Araldite mixture (Electron Microscopy Sciences, Fort Washington, PA, USA). The semi-thin sections (1 mm thick) were examined by light microscopy and stained with toluidine blue. The ultra-thin sections of 70 nm were obtained using a microtome and placed on Cu/Rh grids with Ultracut E (Reichert, Wien, Austria), stained with lead citrate, and observed using an FEI Morgagni 268 D electron microscope (FEI Company, Eindhoven, Netherlands).

To detect the internalization of ASC-exosomes by the cells, exosomes labelled with USPIO nanoparticles, that allowed the visualization by TEM, were used. The NSC-34(G93A) cells were seeded at a density of 2 × 10⁴ with the presence of exosomes-USPIO (0.2 µg/mL, corresponding to 6–8 × 10⁵ particles/mL) in the culture medium for 6 h. After the incubation time, the cells were washed with PBS, trypsinized and centrifuged. For ultrastructural morphology of cells, the pellet was fixed in 2% glutaraldehyde in Sorensen buffer (pH 7.4) for 2 h. The samples were post-fixed in 1% osmium tetroxide (OsO₄) for 2 h, cut, dehydrated in graded concentrations of acetone and embedded in Epon-Araldite mixture (Electron Microscopy sciences, Fort Washington, PA, USA). The semithin sections (1 µm in thickness) were examined by light microscopy (Olympus BX51, Olympus Optical, Hamburg, Germany) and stained with toluidine blue. The ultrathin sections were cut at a 70 nm thickness, placed on Cu/Rh grids with Ultracut E (Reichert, Wien, Austria). TEM images were acquired with a Philips Morgagni TEM operating at 80kV and equipped with a Megaview II camera for digital image acquisition.