Morada soft imaging system

HeLa cells were seeded at 3 × 10⁴ cells per chamber in a Lab-Tek chamber slides of 4 wells (Nalge Nunc International) and treated as indicated in each case. Then, the cells were fixed for 1 h in 3.5% glutaraldehyde at 37°C and postfixed for 1 h in 2% OsO₄ at room temperature. Cellular staining was performed at 4°C for 2 h in 2% uranyl acetate in the dark. Finally, cells were rinsed in sodium phosphate buffer (0.1 mol/L, pH 7.2), dehydrated in ethanol, and infiltrated overnight in Araldite (Durcupan, Fluka). Following polymerization, embedded cultures were detached from the chamber slide and glued to Araldite blocks. Serial semi-thin (1.5 μm) sections were cut with an Ultracut UC-6 (Leica), mounted onto slides and stained with 1% toluidine blue. Selected semi-thin sections were glued (Super Glue, Loctite) to araldite blocks and detached from the glass slide by repeated freezing (in liquid nitrogen) and thawing. Ultrathin (0.07 μm) sections were prepared with the Ultracut and stained with lead citrate. Finally, photomicrographs were obtained under a transmission electron microscope (FEI Tecnai Spirit G2) using a digital camera (Morada, Soft Imaging System, Olympus).

For exosome immunogold labeling for electron microscopy, exosomes were prepared and labeled according to the protocol of Thery et al [41 ]. In brief, isolated exosomes were resuspended in 2% paraformaldehyde (PFA), adsorbed unto nickel Formvar-carbon coated electron microscopy grids (200 mesh), blocked with PBS/5% (w/v) BSA and incubated with antibodies for Her-2 (MSK044, Zytomed Systems, Berlin, Germany), ERα (sc-8002, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Glypican-1 (sc-101827, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. Subsequently grids were washed, blocked and incubated with Donkey Anti-Mouse IgG H&L 10nm Gold antibody (Abcam, Cambridge, United Kingdom). Grids were washed, fixed with 1% glutaraldehyde, contrasted with 4% uranyl acetate and finally embedded in a mixture of 4% uranyl acetate and 2% methyl cellulose.
Grids were visualized on a Morgagni 268 Electron Microscope (FEI, Eindhoven, The Netherlands) and photographed with the Morada Soft Imaging System (Olympus Corporation, Hamburg, Germany).

For immunogold labeling for electron microscopy, exosomes were prepared and labeled according to previous described protocols. In brief, isolated exosomes were resuspended in 2% paraformaldehyde (PFA), adsorbed unto nickel Formvar-carbon coated electron microscopy grids (200 mesh), blocked with PBS/5% (w/v) BSA and incubated with CD30 antibody (DAKO, A/S, Denmark) for 30 min. Subsequently, grids were washed, blocked and incubated with Donkey Anti-Mouse IgG H&L 10 nm Gold antibody (Abcam, Cambridge, United Kingdom). Grids were washed, fixed with 1% glutaraldehyde, contrasted with 4% uranyl acetate and finally embedded in a mixture of 4% uranyl acetate and 2% methyl cellulose. Grids were visualized on a Morgagni 268 Electron Microscope (FEI, Eindhoven, The Netherlands) and photographed with the Morada Soft Imaging System (Olympus Corporation, Hamburg, Germany).

For transmission electron microscopy (TEM) analysis, uninjured (ut) R-stage and NR-stage animals and after 2, 6, 10 and 20 after spinal cord injury (n = 2/3 each point) were anesthetized and immersed (R-stage) or perfused with 0.83 PBS (NR-stage) followed by incubation in 2% PFA and 2.5% glutaraldehyde (EMS, Hatfield, PA). Spinal cords were micro-dissected and post-fixed overnight in the same fixative. Spinal cords were processes as described before [48 (link)]. Briefly, spinal cords at different days post injury were post-fixed in 2% osmium for 2 h, rinsed, dehydrated, and embedded in araldite (Durcupan; Fluka, Buchs, Switzerland). Semi-thin horizontal sections (1.5 mm) were cut with a diamond knife and stained with toluidine blue. To study the cellular response to injury at the different days and stages, ultrathin sections (60–70 nm) were cut with a diamond knife, stained with lead citrate, and examined under a transmission electron microscope (TEM) (Tecnai Spirit G2; FEI, Eindhoven, The Netherlands) by using a digital camera (Morada, Soft Imaging System; Olympus, Tokyo, Japan). Brightness and contrast adjustment of the pictures was performed with Adobe Photoshop (Adobe Systems, San Jose, CA).