Copper grid

To assess the ultrastructure of the obtained EVs, we fixed it in 2.5% glutaraldehyde. After 12 h from the start of fixation, the samples were placed in a 1% OsO₄ solution in phosphate buffer with the addition of sucrose, dehydrated and embedded in Epon-812 (Fluka, Charlotte, NC, USA). Ultrathin sections of 0.1 µm thickness were prepared on a Leica UC7 ultramicrotome (Leica, Wetzlar, Germany) and mounted on copper grids (Sigma). Sections were counterstained with uranyl acetate and lead citrate and then examined using a Hitachi 7700 transmission electron microscope (Hitachi, Tokyo, Japan).

For electron microscopy, ultrathin sections (transverse and longitudinal) mounted on copper grids (Sigma-Aldrich, USA, 200 mesh) were incubated with uranyl acetate and lead citrate for double contrast. Sections were examined with a transmission electron microscope, Jeol 1200 SX (Tokyo, Japan). For immunoelectron microscopy, after dehydration, pieces of spinal cord were embedded in LR-white (Sigma, USA). Ultrathin sections were mounted on nickel grids with formvar film (Sigma-Aldrich, USA, 200 mesh) and blocked with TBS-NGS-BSA-Tx100 (Tris-buffered saline (Tris 0.01 M, NaCl 0.15 M pH = 8.2), normal goat serum 10%, bovine serum albumin 0.2%, and Triton X-100 0.1%) for 1 h. After rinsing in TBS, sections were incubated overnight at 4°C with anti-glial fibrillary acidic protein (GFAP, Santa Cruz, 1 : 150) and anti-human nuclei antigen (HNA, Millipore, 1 : 500) antibodies (Ab) and then 10 nm gold-conjugated secondary Ab (Sigma-Aldrich, USA) for 1 hour at room temperature. For enhanced visualization, silver (Silver Enhancer Kit, Sigma-Aldrich, USA) was deposited on the colloidal gold. Then, sections were double-contrasted with uranyl acetate for 20 min at 60°C and lead citrate for 10 min at room temperature; they were examined by transmission electron microscopy.

A. viridis tissues, dissected from healthy tentacles and from regenerating areas, were fixed for 2 h in 4% glutaraldehyde in a cacodylate buffer (pH 7.4). After several washes in the same buffer, the samples were post-fixed for 1 h with 1% osmium tetroxide in a 0.1M cacodylate buffer (pH 7.4). After serial ethanol dehydration (70%, 90%, and 100%), the specimens were embedded in an Epon-Araldite 812 mixture. For the ultrastructural TEM analyses, ultrathin sections (80 nm in thickness), obtained with a Reichert Ultracut S ultratome (Leica), were placed on copper grids (300 mesh, Sigma-Aldrich, Milan, Italy), counterstained with uranyl acetate and lead citrate, and observed with a Jeol 1010 EX transmission electron microscope (Jeol, Tokyo, Japan). Images were recorded with a MORADA digital camera system (Olympus, Tokyo, Japan).

Since the tissues had been immersed in a solution of unknown composition, sections of the specimens were washed several times in phosphate buffer saline (PBS) buffered formaldehyde before being processed for hematoxylin and eosin (H&E)-histology according to standard procedures. For electron microscopic inspections, small parts of the specimen were transferred into McDowell’s and Trump’s 4F:1G fixative (Sigma-Aldrich, Taufkirchen, Germany) for 19 days (3 changes) and rinsed in PBS pH 7.2 for 7 days (4 changes). Post fixation in 2% OsO₄ (Polysciences, Hirschberg, Germany) in the same buffer for 2 h was performed. Subsequently, tissue specimens were dehydrated in a graded series of alcohol (30–100%), diluted with 5% aqueous solution of uranyl acetate (Polysciences), dehydrated in propylene oxide (Sigma-Aldrich) and embedded in resin Durcupan ACM (Sigma-Aldrich). Ultrathin sections (70 nm thick) obtained using diamond knives in a Reichert Ultra Cut microtome (Reichert, Vienna, Austria) were collected on copper grids (300-mesh) (Sigma-Aldrich). Grids were stained with 2% aqueous uranyl acetate and viewed at 80 kV with a microscope Jeol JEM, 2000 CX (Arishima, Tokyo, Japan) equipped with an Olympus Megaview II digital camera (Olympus Europa SE & Co, Hamburg, Germany).

For TEM investigations, specimens were fixed in 2.5% glutaraldehyde solution (Sigma-Aldrich Chemie GmbH, Germany) buffered with sodium cacodylate buffer (Sigma-Aldrich Chemie GmbH, Germany) at pH = 7.4 for two hours at 4 °C. The samples were postfixed for 1 h in 1% osmium tetroxide solution (Agar Scientific, Stansted, UK) at the same temperature and pH, dehydrated at increasing concentrations of ethanol (50, 70, 90, 96, 99.5%), in an acetone series and embedded in Epon-812 (Fluka Chemie AG, Buchs, Switzerland) according to standard methods. Semithin sections were stained with methylene blue-azur II to select the region of interest for the following procedures. The semithin sections were analysed using a Zeiss Axiophot 2 microscope (Zeiss, Germany). The ultrathin (80 nm) sections were cut on the Reichert Om U3 ultratome with a diamond knife (Diatome Ltd, Biel/Bienne, Switzerland). Sections were mounted on copper grids of mesh size 200 (Sigma-Aldrich Chemie GmbH, Germany) with Perfect Loop (Diatome Ltd., Biel/Bienne, Switzerland) and stained with uranyl acetate (Agar Scientific, Stansted, UK) and lead citrate (Agar Scientific, Stansted, UK) according to standard methods. For TEM, a Philips Tecnai-10 with camera Mega View II was used for viewing and photographing.

Native hADSCs, hADSCs-BFP and hADSCs-IL2 were fixed in 2.5% glutaraldehyde in PBS for 24 h at 4 °C. Post-fixation, cells were treated with 1% osmium tetroxide for 2 h and subsequently dehydrated using ethanol at 30 to 96% v/v concentrations, acetone and then a final treatment in propylene oxide before embedding in Epon 812 resin. After resin polymerization at 37, 45, and 60 °C, samples were cut into ultrathin sections using ultramicrotome (Leica UC7, Leica Biosystems, Wetzlar, Germany). Sections were mounted on copper grids (Sigma-Aldrich, St. Louis, MO, USA, 200 mesh) and contrast agents uranyl acetate and lead citrate were added. Ultrathin sections were examined using a transmission electron microscope (TEM) HT7700 (Hitachi, Tokyo, Japan) at 100 kV.

For electron microscopy, ultrathin sections (transverse and longitudinal) mounted on copper grids (Sigma-Aldrich, USA, 200 mesh) were incubated with uranyl acetate and lead citrate for double contrast. The sections were examined under a transmission electron microscope Jeol 1200 SX (Tokyo, Japan). For immunoelectron microscopy, after dehydration, pieces of the spinal cord were embedded in LR-white (Sigma, USA). Ultrathin sections were mounted on nickel grids with a formvar film (Sigma-Aldrich, USA, 200 mesh) and blocked with TBSNGS-BSA-Tx100 (Tris-buffered saline (Tris 0.01 M, NaCl 0.15 M pH = 8.2), normal goat serum 10%, bovine serum albumin 0.2%, and Triton X-100 0.1%) for 1 h. After rinsing in TBS, the sections were incubated overnight at 4°C with anti-P0 (P0, Santa Cruz, 1 : 150) antibodies and then 10 nm gold-conjugated secondary Ab (Sigma-Aldrich, USA) for 1 hour at room temperature. To enhance visualization, silver (Silver Enhancer Kit, Sigma-Aldrich, USA) was deposited on colloidal gold. Then, the sections were double-contrasted with uranyl acetate for 20 min at 60°C and lead citrate for 10 min at room temperature; they were examined with transmission electron microscopy.