Epoxy resin

Hemocytes were fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde (Thermo Fisher Scientific, Frederick, MD, USA) in a culture medium. Postfixation was carried out with 1% osmium tetroxide (Sigma-Aldrich, St. Louis, MO, USA) in 0.05 M cacodylate buffer (Vekton, Russian Federation), pH 7.4, using a low-temperature automatic water replacement system Leica EM AFS2 (Leica Microsystems, Germany).
The samples were infiltrated with epoxy resin (Agar Scientific Ltd, UK) / acetone (Vekton, Russian Federation) (1:3) for 30 min, followed by resin / acetone (1:2) for 30 min, followed by 1 h with resin / acetone (1:1) under room temperature. The samples were then transferred into the 100% resin; the polymerization was carried out over 24 h at +60°C. Resin blocks were carefully trimmed using a Leica EM UC7 trimmer. Ultrathin slices were collected on the mesh copper grids coated by carbon film (SPI Supplies, West Chester, PA, USA), post-stained with 1% uranyl acetate (Agar Scientific Ltd, UK) in water for 5 min, and with a lead solution for 7 min, and washed in distilled water. The material was then analyzed with a transmission electron microscope JEM– 1400 (Jeol, Japan).

Cells were fixed with 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer containing 5 mM CaCl₂ (pH 7.4), postfixed in 1% osmium tetroxide supplemented with 0.5% potassium hexacyanoferrate tryhidrate and potasssium dichromate in 0.1 M cacodylate for 1H, stained with 2% uranyl acetate in double distilled water for 1H, dehydrated in graded ethanol solutions and embedded in epoxy resin (Agar scientific Ltd., Stansted, UK). Ultrathin sections (70–90 nm) were obtained with an EMUC7 ultramicrotome and were stained with lead citrate and then examined with a Tecnai T12 transmission electron microscope (Thermo Fisher Scientific, The Netherlands). Digital electron micrographs were recorded with a bottom-mounted 4k CMOS camera system (TemCam-F416, TVIPS, Gauting, Germany).

Cells were grown on Thermanox coverslips (Nalgene, Nunc) and fixed with 2% PFA, 2.5% glutaraldehyde in 0.1 M cacodylate. Cells were then secondarily fixed with 1% osmium tetroxide followed by incubation with 1% tannic acid to enhance contrast. Cells were dehydrated using increasing percentages of ethanol before being embedded onto Epoxy resin (Agar scientific, UK) stubs. Coverslips were cured overnight at 65 °C. Ultrathin sections were cut using a diamond knife mounted to a Reichert ultracut S ultramicrotome and sections were collected onto copper grids. Grids were post-stained with drops of lead citrate. Sections were viewed on a FEI Tecnai transmission electron microscope (Eindhoven, The Netherlands) at a working voltage of 80 kV. BSA-gold was prepared as previously described [27 (link)]. For quantification of MVB diameter, MVBs were defined as organelles containing intraluminal vesicles and monomeric rather than flocculated BSA-gold.
Aβ1-42 peptides (Sigma) were incubated at 37 °C for 1 h to induce fibril formation in vitro. Grids were inverted onto the drops of Aβ1-42, negatively stained with 2% uranyl acetate, washed with water and dried on filter paper before being viewed by EM.

We modified the BSE–SEM method, described by Reichelt et al. [10 (link)], to obtain high-resolution images from semi-thin sections of kidney tissue processed for conventional TEM. The kidney fragments were placed in a glass vial for overnight fixation in freshly prepared 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4. The tissue was washed 3 × 10 min with 0.1 M cacodylate buffer, and then, the buffer solution was replaced with 1% osmium tetroxide in cacodylate buffer for 1 h of postfixation. After postfixation, specimens were dehydrated through a series of ascending grades of ethanol concentrations (10% ETOH × 5 min, 50% × 10 min, 70% × 10 min, 90% × 15 min, and finally, 100% × 15 min) and were rinsed with propylene oxide for 15 min. The propylene oxide was replaced with 50% epoxy resin (Agar Scientific Ltd, Stansted, UK.) and 50% propylene oxide mixture (v/v, 1 mL per sample), and the tissue was immersed for 4 h. This mixture was freshly prepared in advance, at room temperature. The intermediate solution was replaced with epoxy resin for 12 h (overnight) for tissue infiltration, and each fragment was gently transferred to the tip of the embedding mould. Moulds were placed in the oven at 60°C for 72 h to obtain polymerised blocks. This critical step required careful control of working temperature (58–60 °C).

After fixation in Karnovsky’s solution, the samples were processed as previously described^{45 (link)}. Ultrastructural organization was evaluated only in fresh controls and tissue obtained from 3 patients soon after warming, which showed higher follicle density. Post-fixation was achieved with 1% osmium tetroxide and contrast was performed with 0.5% uranyl acetate. A solution of 0.1 M cacodylate buffer was used to wash the tissue between steps. Increasing concentrations of ethanol were utilized to dehydrate the tissue before its inclusion in epoxy resin (Agar Scientific, Essex, UK). The blocks were subjected to semi-thin sectioning (3 µm), stained with toluidine blue, and analyzed by light microscopy to localize follicles. Thereafter ultrathin sections (70–90 nm) were obtained and analyzed by TEM (Zeiss, Germany). For TEM evaluation, the morphological characteristics of the stroma, granulosa cells and oocytes, as well as their organelles, basal and plasmatic membranes and nuclear envelope, were taken into account. Shape, morphology, distribution and the electron density of chromatin were also investigated.

The samples were fixed in 2% glutaraldehyde (POCH) and centrifuged (5 min, 50 µf). Contrasting was performed with 2% uranyl acetate (MicroShop, Piaseczno, Poland) (8 h) and 2% osmium tetroxide (Agar Scientific, Stansted, UK) (2 h) in the dark. The material was then passed through an ascending alcohol series (POCH from 30% to 99.8%) and embedded in epoxy resin (Agar Scientific, Stansted, UK). Sections of 60 nm thickness were prepared from the resin blocks using an UltraMicrotome Leica EMUC7 (Leica, Wetzlar, Germany) and placed on copper grids (400 Mesh) with formvar film and carbon coating (Agar Scientific, Stansted, UK). Imaging was performed using a JEOL 1200, (JEOL, Tokyo, Japan) microscope.