Epon resin

Cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M cacodylate buffer, pH 7.4, for 45 min at 4 °C, rinsed in buffer, postfixed in 1% OsO₄ in 0.1 M cacodylate buffer, pH 7.4, dehydrated, and embedded in Epon resin (Agar Scientific). Grids were thoroughly rinsed in distilled water, stained with aqueous 2% uranyl acetate for 20 min. and photographed in a Zeiss EM 900 electron microscope. The number of autophagic vacuoles were counted under the Zeiss EM 900 electron microscope at ×12.000 magnification (48 μm²) for each treatment conditions. Autophagic vacuoles were classified as autophagosomes when met two or more of the following criteria: double membrane, compartments of 0.5 μm in diameter or larger, luminal uncompacted cytosolic material including organelles, absence of ribosomes attached to the cytosolic side of the membrane. We examined 70–100 fields per treatment condition and value are expressed as AVs per field. Finally, data were averaged to median values ± standard deviation (±SD) and used for statistical analysis.

Peripheral blood mononuclear cells were fixed with 3% glutaraldehyde in 0.2 M Sorensen’s phosphate buffer (pH 7.4) for 2–4 h at 4°C, post-fixed in 1% OsO₄ in 0.1 M Millionig’s buffer (pH 7.4) followed by dehydration in 70, 90, 100% alcohol and embedded in epon resin (Agar Scientific, 45359-1EA-F). The blocks were polymerized at 60°C overnight. 50 nm thin sections were stained with aqueous 1% uranyl acetate for 20 min and photographed in a JEM 1400 Plus electron microscope (Jeol Ltd., Tokyo, Japan). Three independent examiners carried out the assessment in 30–35 micrographs from three subjects per group, imaged at 10,000× in a blinded manner and the average number of mitochondria per cell was evaluated. To define the distorted mitochondrial morphology, the following characteristic features such as, individual mitochondrial area, total mitochondrial area per cell (mitochondrial mass), aspect ratio (length of the major axis divided by the length of minor axis of the ellipse equivalent to the mitochondrion), and form factor (perimeter²/4π* Area) were measured using Image J software. Aspect ratio represents the mitochondrial length, while form factor indicates the mitochondrial branching. In addition, degeneration of cristae and matrix were also taken into consideration for calculating the percentage of distorted mitochondria.

Young adult hermaphrodites were processed as previously described (57 (link)). Briefly, worms were fixed in 2.5% glutaraldehyde (Merck) in Sørensen's phosphate buffer (0.1 M, pH 7.4) for 48 h at 4°C, post-fixed in 1% osmium tetroxide (EMS) for 1 h, and dehydrated through an increasing ethanol gradient. Samples were treated with propylene oxide (Sigma) and embedded in EPON resin (Agar Scientific) for 24 h at 60°C. Serial, ultra-thin (90 nm) sections of the worm nose tissue were cut using a Leica EM UC6 Ultramicrotome, collected on copper grids (EMS), stained with 2% uranyl acetate (Agar Scientific) for 20 min followed by 3% lead citrate (LabTech) for 5 min, and imaged on a Tecnai 12 (FEI software) with an acceleration voltage of 120 kV.

Retina samples (6 control and 6 diabetic retinas) taken from the central part of the left eye were dehydrated in ascending concentrations of ethanol, and were embedded in Epon resin (Agar Scientific, Essex, UK). Embedded capsules were polymerized with ultraviolet light (360–365 nm) overnight at 25 °C in a TAAB ultraviolet chamber [27 (link)]. Semi-thin sections were cut after the blocks were trimmed with a razor blade. Ultra-thin sections (95 nm each) were also made and were mounted onto carbon-formvar-coated 200-mesh Nickel grids using a Reichert Ultracuts ultramicrotome (Leica, Microsystems GmbH, Vienna, Austria). Contrast staining was performed with 2% uranyl acetate and lead citrate, for 15 and 7 min, respectively [27 (link)]. The sections were viewed with a Philips CM10 transmission electron microscope (Philips, Amsterdam, The Netherlands).

To assess cell morphology by electron microscope, the treated cells were fixed in 3% glutaraldehyde, post-fixed in 1% osmium tetroxide solution, dehydrated with acetone, and embedded in Epon resin (Agar Scientific, Stansted, UK). Ultrathin sections were prepared with an Ultracut microtome (Leica, Oskar-Barnack, Germany) and then stained with 4% uranyl acetate and lead citrate. The sections were examined using a JEM-100cxII electron microscope (JEM-1010, JEOL, Tokyo akishima, Japan).

Small samples of muscle tissue of each patient were fixed in 4% paraformaldehyde, postfixed in 2% osmium tetroxide, and dehydrated by a series of incubations in 30%, 50%, and 70% ethanol. Samples were then embedded in EPON resin (Agar Scientific, Stansted Essex CM24 8GF United Kingdom) [17 (link)], for morphological ultrastructural analysis, and in LR-White resin (Agar Scientific, Stansted Essex CM24 8GF United Kingdom), for ultrastructural immunohistochemistry. Tissues were cut [18 ] and stained with heavy metals solutions as described by Reynolds [19 (link)].