Epoxy resin

To further observe the effect of miR-222 on sperm, ultrathin sections of sperm were made after coincubation with SPEVs. One group was coincubated with SPEVs transfected with the miR-222 mimic, and the other group was coincubated with SPEVs transfected with the miR-222 inhibitor.
The sperm samples were centrifuged at 17°C and 800 × g for 5 min, and the supernatant was discarded. Then, the samples were placed in a fixative solution of pH = 7.2 [a mixture of 2% PFA (Alfa Aesar, United States) and 2.5% glutaraldehyde (SPT Supply, United States)] and placed on ice for approximately 1 h. Then, the samples were fixed in 1% osmic acid on ice for 1 h. Next, 1% uranyl acetate (Electron Microscope China, China) was used for staining. The samples were washed with PBS three times after each step. The samples were then dehydrated with different concentrations of alcohol and embedded in epoxy resin (Electron Microscopy Sciences, United States). Finally, an ultrathin slicer (EMUC7, Leica, Germany) was used to slice the blocks at a thickness of 70 nm. After cutting, the sections were imaged by electron microscopy (H-7650B, Tokyo, Japan).

To evaluate skeletal muscle fine sub-cellular ultrastructural features, CB1^+/+ and CB1^−/− skeletal muscle were minced in small pieces and fixed overnight (O.N.) in glutaraldehyde 2.5% (Electron Microscopy Science, Hatfield, PA, USA) in 0.1 M cacodylate buffer (pH 7.4) at 4 °C. Fixed samples were then rinsed with 0.1 M cacodylate buffer for at least 1 h, post-fixed with 1% osmium tetroxide (OsO4) in 0.1 M cacodylate buffer (Electron Microscopy Science, Hatfield, PA, USA), dehydrated in ethanol (Sigma-Aldrich, Milano, Italy, EU), and embedded in epoxy resin (Electron Microscopy Science, Hatfield, PA, USA). Ultrathin sections (60 nm), obtained using an UC6 ultramicrotome (Leica, Wetzlar, Germany, EU) equipped with a diamond knife (DiATOME US, Hatfield, PA, USA), were placed on copper grids (Electron Microscopy Science, Hatfield, PA, USA). Ultrathin sections were then treated with Uranyl Acetate Replacement stain (UAR-Electron Microscopy Science, Hatfield, PA, USA) and contrasted with lead citrate (Sigma-Aldrich, Milano, Italy, EU). Samples were studied using a 100 kV transmission electron microscope EM208S PHILIPS (FEI—Thermo Fisher, Waltham, MA, USA) equipped with the acquisition system Megaview III SIS camera (Olympus/EMSIS) and iTEM3/Radius software, version 2.1.

On day 12, the cells were dissociated with 0.25% trypsin at 37 °C for 3–5 min, and the digestion was stopped with a culture medium containing FBS. The cells were transferred into a 10 mL tube, centrifuged at 1200 rpm for 8 min. They were washed with PBS (4 °C) and then centrifuged at 1200 rpm for a further 8 min. After discarding the supernatant, 2.5% glutaraldehyde in 0.1 M phosphate buffer was added to the cells and left to fix at 4 °C overnight. The cells were rinsed three times with 0.1 M phosphate buffer (pH 7.2) for 15 min each, followed by post-fixation with 1% Osmium tetroxide (OsO₄) prepared in dH₂O. Fixed samples were dehydrated, embedded in epoxy resin (Electron Microscopy Sciences), and polymerized at 60 °C for 24 h. The blocks were ultra-thin-sectioned at 60 nm with a diamond knife using an RMC ultra-microtome. The sections were placed on copper grids and stained with 2% uranyl acetate at room temperature for 15 min and then rinsed with distilled water, followed by secondary staining with lead stain solution (SPI-CHEM) at room temperature for 15 min. The grids were observed under a TEM (FEI Model TECNAI G2 20S-TWIN) at an acceleration voltage of 120 kV.

TA muscle tissues were fixed in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer for 1 h. Fixed muscle fibers were stained with α-bungarotoxin and dissected to isolate regions of muscles containing NMJs. Muscles were washed in 0.1 M sodium cacodylate buffer and then post-fixed in 1% osmium tetroxide for 1 h in the dark. After rinsing with sodium cacodylate, the samples were dehydrated in increasing concentrations of acetone (50% for 10 min, 75% for 10 min, 90% for 10 min, 95% for 10 min, 100% for 4 x 10 min). Muscles were transitioned to epoxy resin (Electron Microscopy Sciences, Hatfield, IL) by rotating the samples overnight in a 50:50 solution of epoxy:acetone. The samples were then immersed in fresh 100% resin several times throughout the next day and baked at 65°C overnight. Ultra-thin cross-sections were collected using a Leica EM-UC6 ultramicrosome (Buffalo Grove, IL) and stained for contrast with uranyl acetate and lead citrate. Samples were viewed using an FEI Tecnai T-12 electron microscope (Delmont, PA) with a Hamamatsu digital camera (Bridgewater, N.J).

For ultrastructural studies, organ samples from 15 mice (five from the control group and five from the groups infected with both DENV2 strains) euthanized at 72 hpi were processed as described by Barreto-Vieira [56 ]. Briefly, samples were fixed by immersion in glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) at 2% diluted in sodium cacodylate buffer (0.2 M, pH 7.2), cleaved into smaller fragments (~1 mm³), post-fixed in 1% osmium tetroxide and dehydrated in increasing concentrations of acetone. Subsequently, the samples were embedded in epoxy resin (Electron Microscopy Sciences). Ultra-thin sections (50–70 nm thick) were obtained with the aid of an ultramicrotome (Leica, Wetzlar, Germany), placed on copper grids and counterstained with uranyl acetate and lead citrate. Finally, the samples were observed under the Hitachi HT 7800 TEM (Hitachi, Tokyo, Japan).