Epoxy resin

EM of PERV-C-HA cells was performed according to standard procedures as described previously [21 (link)]. Briefly, cells were fixed with 2.5% glutaraldehyde (Sigma‐Aldrich, USA) in culture medium for 45 min at room temperature. Cells were washed two times with PBS. Subsequently, sample preparation was performed in the electron microscopy facility at Paul-Ehrlich Institut. Cells were scraped off the culture flask and gently mixed with 2% warm liquid agarose. After cooling and gelling, small agarose blocks containing the cells were cut and post‐fixed with 2% osmium tetroxide (Sigma‐Aldrich, USA) in PBS and treated with 1% tannic acid (Carl Roth, Germany). Cells were gradually dehydrated in a series of ethanol baths and embedded in Epoxy resin (Sigma‐Aldrich, USA). Polymerization lasted for 48 h at 60 °C, afterwards ultrathin sections were cut and stained with 2% uranyl acetate (Merck, Germany) for 15 min followed by 2% lead citrate (Serva, Germany) for 5 min. Sections were examined in a Jeol JEM 1400 Flash electron microscope using electron filtering (ESI) mode.

The cells were fixed in 2.5% glutaraldehyde and embedded with epoxy resin (Sigma-Aldrich, USA). The sample sections were stained with lead citrate (Sigma-Aldrich, USA) and assessed under the electron microscope (JEM-2100F, Hitachi, Japan).

Tissue samples were fixed overnight at 4 °C in 2.5% glutaraldehyde (Sigma-Aldrich) and 2% paraformaldehyde (PFA; Polysciences) in 0.1 mol l⁻¹ sodium cacodylate buffer pH 7.4. The samples were post-fixed with 1% osmium tetroxide 1% (EMS) in 0.1 mol l⁻¹ sodium cacodylate buffer for 1 h at 4 °C. After three water washes, samples were dehydrated in a graded ethanol series and embedded in epoxy resin (Sigma-Aldrich). Ultrathin sections (60–70 nm) were obtained with an Ultrotome V (LKB) ultramicrotome, counterstained with uranyl acetate and lead citrate and viewed with a Tecnai G² (FEI) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System) digital camera.

Organoids at day 8 of the proliferative stage were released from the 1.5-kPa hydrogel and fixed in 2.5% glutaraldehyde and 2% PFA diluted in 0.1 M phosphate buffer for 8 hours. After washing in PB and dehydration in an ethanol gradient, the samples were embedded in epoxy resin (Sigma-Aldrich). Samples were sectioned at a thickness of 50 nm and mounted on copper grids. After staining with 4% uranyl acetate for 5 min, the sections were stained with 0.4% lead citrate for 1 min. The ultrastructure of the sections was visualized with a transmission electron microscope (CM120, Philips).

U87-MG, MO3.13 cells, and primary fibroblasts cultured from skin biopsies of healthy subjects and ADLD patients were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, for 2 h at 4 °C and then, post fixed in 1% OsO₄ in 0.1 M cacodylate buffer, for 30 min at room temperature. After few washes in 0.15 M cacodylate buffer, samples were dehydrated through graded acetone solutions and embedded in Epoxy resin (Sigma-Aldrich). Sections of 100 nm each were collected in nickel grids, counterstained with 3% uranyl acetate and 1% lead citrate and observed by Philips CM10 TEM (FEI Company, Eindhoven, The Netherlands), at an accelerating voltage of 80 kV. Images were recorded by Megaview III digital camera (FEI Company).

The cuticle of Ae. aegypti in the abdomen were dissected from the native larvae of the first to fourth instar (sampling immediately after molting) (n = 10), and RNAi-treated fourth-instar larvae (n = 10). Normal cuticles of the wild larvae groups in the same instar were used as a blank control. Specimens were fixed in 2.5% glutaraldehyde (Kemiou, Tianjin, China) in phosphate buffer (0.1 M, pH 7.0) overnight, and samples were washed 3 times for 5 min with phosphate buffer. Specimens were further fixed with 1% osmium tetroxide buffer in phosphate buffer (0.1 M, pH 7.0) at 4 °C for 1–3 h. After fixation, specimens were washed 3 times with phosphate buffer (0.1 M, pH 7.0) for 5 min. The samples were gradually dehydrated with 30, 50, 70, 90, 95 and 100% ethanol for 5 to 10 min at each step; the 100% ethanol step was repeated 2 to 3 times to ensure complete dehydration. After dehydration, the samples were embedded in epoxy resin (Sigma-Aldrich, St. Louis, USA). The specimens were sliced with an ultramicrotome (Leica UC6; Leica, Vienna, Austria). The sections were stained with uranyl acetate (Syntechem, Changzhou, China) and lead citrate (Acros, Shanghai, China) and observed using an electron microscope (JEM1200EX; Jeol, Tokyo, Japan).