Epon 812 resin

Adult specimens of Lineus ruber were collected near Bergen, Norway (Fanafjord; GPS coordinates: 60.251845 N, 5.320947 E). The animals had dark red coloration with wide pigment-free areas in the terminal part of the head. Animals were kept in the laboratory in filtered seawater at 14 °C with a daytime cycle: 13 h of sunshine and 11 h of darkness. Collection of egg masses and desired developmental stages and animal fixation as well as antibody, nuclear, and EdU stainings followed the already established protocols [38 (link)].
Specimens for TEM investigation were fixed in 4% PFA in PBS, rinsed in the same buffer, postfixed in 1% OsO4 diluted in PBS for 120 min at 4 °C, rinsed again, and dehydrated in graded ethanol/acetone series. The samples were embedded in Epon 812 resin (Sigma Aldrich) and cut to semi- and ultrathin sections with a diamond knife (Diatome Histo Jumbo) using ultramicrotome Leica EM UC6. The ultrathin cross-sections of cerebral organ were placed on formvar-covered (Fluka) single slot copper grids and stained with 1% uranyl acetate and lead citrate.

Roots from 5-, 10-, and 15-day-old plants, in vitro grown, were first fixed with 3% (v/v) glutaraldehyde in 0.025 M cacodylate buffer (pH 7.3) at room temperature. After washing, the samples were post-fixed with 1% OsO4 in the same buffer. The samples were then dehydrated in a methanol series (30, 50, 70, and 100%) at room temperature. The samples were acetylated and methylated with a freshly prepared 5:1 (v/v) methanol/acetic anhydride mixture at 25°C. Samples were then washed in pure methanol and embedded in Epon 812 resin (Sigma). Ultrathin sectioning was performed on an ultramicrotome (Leica Ultracut), and counterstained with uranyl acetate and lead citrate before being observed using a 7500 Hitachi TEM.

L. amazonensis promastigotes treated with IC₅₀ for (-)-5-demethoxygrandisin B and incubated for 24 h at 26 °C were collected by centrifugation at 5000 rpm for 5 min. The parasites underwent fixation for an overnight duration within a solution comprising 2.5% glutaraldehyde (Sigma, St Louis, MO, USA) immersed in 0.1 M sodium cacodylate buffer (pH 7.2). After this, a sequence of three washes utilizing 0.1 M sodium cacodylate buffer followed. Subsequently, a post-fixation procedure was carried out through immersion in a solution containing 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 5 mM calcium chloride. Following further washing steps in 0.1 M sodium cacodylate buffer, the parasites underwent dehydration through a graduated acetone series before being embedded within EPON 812 resin (Sigma, St Louis, MO, USA). Ultrathin sections, spanning 100 nm, were then attained via Sorvall MT 2-B (Porter Blum) ultramicrotome (Sorvall, Newtown, CT, USA). These sections were subsequently stained with a 5% uranyl acetate aqueous solution and lead citrate (comprising 1.33% lead nitrate and 1.76% sodium citrate) prior to observation under a transmission electron microscope JEM-1011 (JEOL, Tokyo, Japan) operating at 80 kV [25 (link)].

Mitochondrial injury (i.e., deformation of mitochondrial cristae or inner mitochondrial membrane disruption) and autophagosome formation (i.e., cytoplasmic components enclosed in double-membraned vesicles) were assayed using transmission electron microscopy analysis, as previously reported [10 (link),53 (link)]. Harvested liver tissues were fixed, washed, and fixed again in 2% osmium tetroxide and embedded in Epon 812 resin (all from Sigma-Aldrich). The grids containing sections were stained with 2% uranyl acetate and 0.2% lead acetate (both from Sigma-Aldrich) followed by ultrathin sectioning (60–70 nm). The sections were examined with a transmission electron microscope (Hitachi HT-7700; Hitachi, Tokyo, Japan). Five random fields (2 μm²) were selected to count the numbers of intact mitochondria, as well as autophagosomes, in each group by a pathologist blinded to the treatment.

TEM of maize pollen ultrastructure was performed in accordance with previously described methods [24 (link)] with a slight modification. Freshly collected pollen grains were fixed at 4 °C in 2.5% glutaraldehyde (in 0.1 M PBS, pH 7.4) overnight and then in 1% osmic acid (0.1 M PBS, pH 7.4) for 6 h. After fixation, the samples were washed several times in PBS and dehydrated in a series of increasing ethanol concentrations (30, 50, 70, and 90% for 15 min each) and then in acetone twice (for 15 min each). The dehydrated samples were embedded in a mixture of acetone and Epon812 resin (1:1, 1:3, for 30 min each; Sigma-Aldrich) and then in pure Epon812 resin for 2 h. The embedded blocks were polymerized at 37 °C and 45 °C for 12 h each, followed by 60 °C for 48 h. Sections were cut using the Leica EM UC7 ultramicrotome with a diamond knife. After staining with uranyl acetate and lead citrate, the sections were observed using a HT7700 microscope (Hitachi, Ibaraki, Japan).

Overnight culture of T. thermophilus HB8 cells was used to inoculate 20 mL of the 162 medium at 60°C and grown until exponential growth was reached. The cells were centrifuged, washed and resuspended in 20 mM Tris–HCl, pH 8.0 to a final concentration of ~10⁷ cells in a volume of 500 μL. The bacteria were incubated at 60°C for 10 min with PhiKo endolysin (6.25 μg/mL). As a control, buffer without protein was used. After incubation, bacteria were centrifuged and the pellet was fixed with 2.5% glutaraldehyde and post-fixed with 1% osmium tetroxide (Polysciences Inc.). Samples were dehydrated with ethanol and embedded in Epon 812 resin (Sigma-Aldrich). Ultrathin sections (60 nm) were obtained with ultramicrotome (Leica UC7), and stained with lead citrate and uranyl acetate (Sigma-Aldrich). TEM analyses were performed at the Laboratory of Electron Microscopy (Faculty of Biology, University of Gdansk, Poland). Bacteria were visualized using Tecnai Spirit BioTwin electron microscope (FEI Company).

Promastigote forms of L. amazonensis were treated with IC₅₀ for ACFF or luteolin, for 24 h. Non-treated parasites were used as a negative control. After 24 h-incubation at 26°C promastigotes were collected by centrifugation at 5,000 rpm for 5 min. The parasites were fixed with 2.5% glutaraldehyde (Sigma, St Louis, MO, United States) in 0.1 M sodium cacodylate buffer, pH 7.2, overnight. Then, parasites were washed three times with 0.1 M sodium cacodylate buffer and post-fixed in a solution containing 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 5 mM calcium chloride, washed in 0.1 M sodium cacodylate buffer, dehydrated in graded acetone, and embedded in EPON 812 resin (Sigma, St Louis, MO, United States). Ultrathin sections were obtained from 100 nm cuts in Sorvall MT 2-B (Porter Blum) ultramicrotome (Sorvall, Newtown, CT, United States) stained with 5% uranyl acetate aqueous solution and lead citrate (1.33% lead nitrate and 1.76% sodium citrate), and examined in a transmission electron microscope JEM-1011 (JEOL, Tokyo, Japan) operating at 80 kV (Mondêgo-Oliveira et al., 2021 (link)).