Uranyless solution

Transmission electron microscopy was carried out as detailed in previously published protocols^{17 (link),37 (link)}. Epon 812 resin-embedded human testicular specimens were cut with a RMC MT-X ultramicrotome (EMME3, Milan, Italy) and semithin sections (2 μm thick) were stained with a toluidine blue solution in 0.1 M borate buffer and examined under a light microscope. Ultrathin sections (~70 nm thick) of the selected areas were subsequently obtained using a diamond knife and stained with ready-to-use UranyLess solution (Electron Microscopy Sciences, Foster City, CA, USA) followed by an alkaline bismuth subnitrate solution. The stained ultrathin sections were observed and photographed by a high resolution digital camera connected to a JEOL JEM-1010 electron microscope (Jeol, Tokyo, Japan)^{17 (link),37 (link)}.

Shape and surface morphology of NP–XL were analyzed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). A suspension NP–XL in distilled water was dropped onto the surface of a formvar-coated copper grid (Gilder Grid Square 200 Mesh), followed by the evaporation of the solvent to prepare sample for TEM. Then, 10 µL of UranyLess solution (Electron Microscopy Sciences, Hatfield, PA, USA) was also dropped onto the surface of a formvar-coated copper grid and incubated for 20 s to enhance the contrast of the TEM images. The grid was then washed twice in deionized water. To prepare the sample for SEM, a drop of NP–XL water suspension (1 mg/mL) was placed on a double-sided carbon tape, respectively, and dried for 30 min. Samples were coated with a gold layer. TEM analysis was performed on a JEM-1400 microscope at 120 kV (JEOL, Tokyo, Japan). SEM analysis was performed on a JSM-6510LV microscope (JEOL, Tokyo, Japan).

Cells were prefixed in a 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for one day at 4°C. The glutaraldehyde-fixed cells were washed three times in 0.1 M phosphate buffer (pH 7.0) and post-fixed in 1% (W/V) OsO4 for 1 h at 4°C. The fixed cells were then rinsed three times with distilled water. Dehydration was carried out at 4°C using a graded ethanol series of 50%, 60%, 70%, 80%, and 90% for 10 min each and three times with 10 min changes of pure ethanol. Pellets were then brought to room temperature and transferred through propylene oxide two times for 20 min each with 50% and 75% Spurr’s embedding resin [12 (link), 23 (link)] in propylene oxide for 1 h each and 100% overnight. The following day, pellets were transferred to new pure resin and polymerized at 70°C. Blocks were thin-sectioned on a PT-X ultramicrotome (RMC Products, Boeckeler Instruments, USA). Sections of 70 nm thickness were collected on slot copper grids, stained with UranyLess solution (Electron Microscopy Sciences, USA) and Reynold’s lead citrate [12 (link), 24 (link)] and observed and photographed using a JEM-2100F transmission electron microscope operated at 120 kV (Jeol, Japan).