Embed 812 kit

HeLa cells were grown on ACLAR disks and transfected with either NL4-3 or NL4-3(NC–Fluo-SP2) vectors. Cells were fixed in 2.5% glutaraldehyde plus 1% paraformaldehyde in 0.1 M cacodylic buffer for 30 min and then embedded in resin using an Embed 812 kit (Electron Microscopy Sciences, Hatfield, PA, USA) and sectioned at 80 nm with a diamond knife (Diatome) using a Leica EM UC6 (Leica Microsystems, Wetzlar, Germany). Sections were visualized using a JEM 1400 Plus electron microscope (JEOL, Tokyo, Japan) at 120 kV.

For observations in electron microscopy, cultures containing 40 mg·L⁻¹ and 100 mg·L⁻¹ of selenate, as well as untreated culture, were cultivated in batch for 240 h. The algal cells were then collected from each culture, washed with culture medium, and collected by centrifugation (2500 rpm, 5 min). The algal cells were fixed with 1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at 4°C. The cells were then washed three times (5 min each one) using the same buffer. The samples were postfixed with 1% osmium tetroxide in 0.2 M cacodylate buffer at 4°C for 1 h. Samples were washed with the same buffer, dehydrated in a graded ethanol series, and embedded in Epon 812 (EMbed 812 Kit; Electron Microscopy Science, Hatfield, PA, USA). Ultrathin sections of 80–90 nm obtained by an ultramicrotome (UCT, Leica, Wetzlar, Germany) and placed on nickel grids were stained with aqueous 1% (w/v) uranyl acetate and lead citrate. Transmission electron micrographs were observed with a JEM 1011 (JEOL Ltd., Tokyo, Japan) electron microscope using an accelerating voltage of 80 kV. Several photographs of entire cells and of local detailed structures were taken at random, analyzed, and compared to investigate selenium effect in the different subcellular structures of Chlorella sorokiniana. All chemicals used for histological preparation were purchased from Electron Microscopy Sciences.

Cells were infected with HSV-1 (MOI = 3) and harvested at the times post-infection as indicated in the figures. Cells were fixed for 1 h at 25° in 2.5% glutaraldehyde and then incubated with 1% osmium tetroxide (OsO₄), dehydrated in a gradient of ethanol (30–100%) and embedded with an Embed 812 kit (Electron Microscopy Sciences, Fort Washington, PA, USA). Ultrathin sections (60–80 nm) were obtained, mounted on copper grids (200 mesh), and stained with 2% (wt/vol) phosphotungstic acid (PTA; pH 6.8). Observations of the grids were made using a Tecnai transmission electron microscope (FEI) operated at 200 kV. Additionally, in order to reflect the extent of GA damage, we measured the Golgi cisternae lumen width (μm) in mock- and HSV-1-infected cells transfected with pcDNA3.1 or pcDNA3.1-GM130. We quantified the maximum length of GA cisternae (designated as maximum luminal width) by measuring the cisternae diameters assuming circular structures. Thirty cells for each condition and 3–5 GA cisternae for each cell were analyzed by electron microscopy. Each image was measured with ImageJ software (National Institutes of Health; Martin et al., 2017 (link)).

The cells were washed three times in 0.1 M cacodylate buffer, fixed in 2.5% (v/v) glutaraldehyde diluted in 0.1 M cacodylate buffer (pH 7.4) and post-fixed in 2% (v/v) osmium tetroxide. The cells were then dehydrated in a gradient of alcohol solutions and embedded in EMbed 812 kit (Electron Microscopy Sciences; Hatfield, PA, USA). Ultrathin sections (60–70 nm) were not counterstained to optimize their observation with a JEOL JEM1400 electron microscope (JEOL; Tokyo, Japan) at 80 kV. Images were obtained with a Megaview III camera and iTEM Five software (Soft Imaging System; Münster, Germany).

Mice were perfused with 4% glutaraldehyde in 0.1 M sodium cacodylate buffer and brains were postfixed overnight and rinsed. Coronal vibrotome sections at 200 were treated with 1% osmium tetroxide, 1.5% potassium ferracyanide in 0.1 M cacodylate buffer, dehydrated in ethanol solutions, stained en bloc in 3% uranyl acetate in 70% ethanol, and then further dehydrated in ethanol followed by propylene oxide. Tissue was infiltrated in resin (Embed 812 kit; Electron Microscopy Sciences) and then embedded. The CA1 sections were cut, and random images were acquired through the stratum radiatum. At least ten images were analyzed per sample. ImageJ was used to count the number of synapses and measure the length of the post-synaptic density.

Platelet pellets were fixed by adding 2.5% glutaraldehyde (w/v) and 1.25% (w/v) formaldehyde in 0.1 M phosphate buffer for 1 h at 4 °C. After carefully removing the fixative, platelets were embedded in 2% (w/v) agarose and allowed to clot at 4 °C. The clotted specimens were cut and cubes of approximately 1-mm were post-fixed in 1% (v/v) osmium tetroxide and 1.5% potassium ferrocyanide (w/v) in 0.1 M phosphate buffer for 1 h at 4 °C. After washing, specimens were stained with 1% (v/v) uranyl acetate for 1 h at room temperature in the dark. Following staining, the specimen was passed through ascending gradual series of ethanol concentrations, starting at 30% for 10 min, followed by 50% for 10 min, 70% for 10 min, 90% for 10 min, and 100% for 10 min, three washes per cycle, to complete specimen dehydration. Subsequently, infiltration was initiated by submerging the specimen in a series of EPON-resin (EMbed 812 Kit, Electron Microscopy Sciences, Hatfield, PA, USA) mixtures at different concentrations to remove ethanol. After infiltration was complete, the specimen was embedded with 100% resin overnight in an oven at 60 °C for polymerization. Ultrasections of approximately 0.8-μm-thickness were cut and examined in a Tecnai^TM G² Spirit BioTWIN (FEI Company, Hillsboro, OR, USA) at 120 kV. Digital images were captured using an Olympus-SIS Veleta digital charge coupled camera.