200 mesh copper grid

Cell pellets were fixed in 2% glutaraldehyde, 2% paraformaldehyde in 0.1 M Sorensen’s phosphate buffer, pH7.2. Samples were post fixed in 1% osmium tetroxide in water, dehydrated, and embedded. 60 to 90 nanometer sections were placed on 200 mesh copper grids (Ted Pella). Sections were stained with 2% Uranyl Acetate and Reynolds’ lead citrate for 5 min each step. Sections were examined with a Technai G2 Transmission Electron Microscope (FEI) operated at 80Kv with an AMT camera for digital imaging.

Testes fragments were fixed by immersion in 4% glutaraldehyde (EMS, USA). Smaller pieces (1–2 mm thickness) were obtained and postfixed in reduced osmium (1% OsO4 and 1.5% potassium ferrocyanide in distilled water) for 90 min, dehydrated in ethanol, and embedded in Araldite epoxy resin. Ultrathin sections (60 nm thick) were obtained using a diamond knife on a Leica EM UC6 ultramicrotome (Leica Microsystems) and mounted on 200 mesh copper grids (Ted Pella). The ultrathin sections were stained with lead citrate (Merck, USA) and analyzed using a transmission electron microscope (Tecnai G2-12 Thermo Fisher Scientific/FEI, USA). The viral particle was defined when presenting a round to oval shape, a membrane, an electron-dense interior, and within a membrane compartment [51 (link)].

For conventional TEM, isolated eosinophils were prepared as before (Melo et al., 2005a (link), 2009 (link)). Cells were fixed in a mixture of freshly prepared aldehydes [1% paraformaldehyde (PFO) and 1.25% glutaraldehyde] in 0.1 M sodium cacodylate buffer (final concentration) for 1 h at RT, embedded in 2% agar and kept at 4°C for further processing. Agar pellets containing eosinophils were post-fixed in 1% osmium tetroxide in sym-collidine buffer, pH 7.4, for 2 h at RT. After washing with sodium maleate buffer, pH 5.2, pellets were stained en bloc in 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0 for 2 h at RT and washed in the same buffer as before prior to dehydration in graded ethanols and infiltration and embedding with a propylene oxide-Epon sequence (Eponate 12 Resin; Ted Pella, Redding, CA). Sections were mounted on uncoated 200-mesh copper grids (Ted Pella) before staining with lead citrate and viewed with a transmission electron microscope (CM 10; Philips, Eindhoven, The Netherlands) at 60 KV.

The morphology of liposomes under the presence of peptide/dendrimer complexes was examined by transmission electron microscopy JEOL-10 (Japan). Dendriplexes were added to liposomes suspended in the 10 mM Na-phosphate buffer at a molar ratio of 1:25 (dendriplex:lipid). The resultant complexes were placed on the carbon surface of a 200-mesh copper grid (Ted Pella, Inc., USA) for 10 min and drained with blotting paper. The samples were stained with 2% (w/v) uranyl acetate for 2 min and dried. A magnification of 50,000–100,000× proved best for examining liposomes interacting with peptide/dendrimer complexes.