Orius200 sc digital camera

After 24h treatment with vehicle, TRB or LUR, cells were washed three times with sterile PBS, and subsequently fixed with Permanox®, in a chamber slide with a mix that contained 4% formaldehyde, 2% glutaraldehyde in cacodylate buffer, for 1h. After washing with the fixing solution, samples were fixed with 1% osmium tetroxide for 1h. Samples were contrasted with 0.5% aqueous uranyl acetate (pre-embedding). Subsequently, samples were dehydrated by adding increasing ethanol concentrations (30, 50,70, 95, 100%) and were gradually included in epoxy resin, to do so, samples were exposed to these solutions; ethanol-epoxy resin (2:1, 1:1, 1:2). To polymerize the epoxy resin, samples are incubated at 60°C for two days, once the epoxy resin is polymerized and dry, 60 nm ultrathin slides are cut with Leica ultramicrotome, samples are located in the grid. Finally, the grid and samples are treated with 2% uranyl acetate and Reynolds’ lead (post-embedding contrast). Ultrastructural studies were conducted using a Transmission Electron Microscope Jeol Jem 1010 equipped with a Gatan Orius200 SC digital camera with an acquisition tension of 80 kV.

Transmission electron microscopy (TEM) was performed as previously described [16 (link)]. Immediately after excision, LV samples were fixed in 4% formahaldehyde: 1% glutaraldehyde in cacodylate buffer, and postfixed in 1% osmium tetroxide. Tissues were then washed in PBS, dehydrated through graded alcohols followed by acetone, and then infiltrated with Durcupan ACM Fluka resin and polymerized at 60 °C for 48 h. Blocks were cut with a Leica ultracut UCT ultramicrotome (Leica, Heerbrugg, Switzerland), and Sects. (60–70 nm) were mounted onto 200-mesh grids. Sections were stained with a 2% solution of aqueous uranyl acetate for 10 min, followed by lead citrate staining for 10 min. Stained sections were viewed with a JEOL JEM-1010 transmission electron microscope (Tokyo, Japan) operating at 80 kV through 6000 × , 10,000 × , and 40,000 × objectives. Images were acquired with a GATAN Orius 200SC digital camera. Mitochondrial morphometry was analyzed using ImageJ (National Institutes of Health).

For negative staining, 5 μL of the purified fractions resuspended in 2% PFA at a concentration of 1 × 10¹¹ particles/mL were each deposited on a parafilm layer. A grid coated with formvar/charcoal was placed over each drop, and the sEVs were allowed to adsorb for 20 minutes. Then they were washed over 5 drops of 100 μL of PBS for 1 minute each, and subsequently each grid was placed over a drop of 50 μL of 1% glutaraldehyde in PBS and fixed for 5 minutes. After this, the grids were washed over 8 drops of 100 μL of distilled water for 2 minutes each and placed on a drop of 50 μL of uranyl-oxalate, contrasted for 5 minutes, and left to dry. Finally, the grids were placed on a drop of 50 μL of methylcellulose–uranyl acetate for 10 minutes on ice and allowed to dry at room temperature. Visualization of the grids was performed on a JEOL JEM1010 transmission electron microscope (100 kV). Images were recorded with a Gatan Orius 200 SC digital camera.