Epoxy embedding medium

The HCECs’ ultrastructural change, after MXF exposure, was investigated by transmission electron microscopy (TEM). The HCECs were treated with various concentrations of MXF (0, 0.25, 0.5, and 1.0 mg/ml) for 48 h, and then the cells were fixed in 3.7% paraformaldehyde (Sigma–Aldrich, St. Louis, MO, USA) and 2.5% glutaraldehyde (Sigma–Aldrich) in 0.1 M phosphate buffer (PB; pH 7.6) overnight. After washing in 0.1 M PB, the HCECs were fixed in 1% of osmium tetroxide in the same buffer for 1 h. Following the dehydration of the cells with a series of graded EtOH (Merck, Kenilworth, NJ, USA), HCECs were embedded in an epoxy embedding medium (Sigma–Aldrich). Then, polymerization was performed at 60 °C for three days. The ultrathin Sects. (60–70 nm) of the samples were obtained and examined under TEM (JEM-1010; JEOL, Tokyo, Japan), operating at 60 kV. The images were recorded by a charge-coupled device camera (SC1000; Gatan, Warrendale, PA, USA). The length of the electron micrograph was measured using the GMS software (Gatan).

For ultrastructural analysis, cells were plated in a Chamber Slide™ system and fixed in 0.5% glutaraldehyde and 2% paraformaldehyde, 0.1 M cacodylate buffer, pH 7.4 for 1 h, then post fixed in 1% osmium tetroxide in the same buffer for 45 min, in the dark. Samples were washed for 30 min and contrasted en bloc with 1% uranyl acetate in the dark, then gradually dehydrated in ethanol. All the steps of the above procedure were performed at 4°C. Cells were infiltrated with a mixture of ethanol and Epoxy Embedding Medium (Sigma-Aldrich™, Cat# 45359-1EA-F), then embedded in the same resin, allowing specimens to polymerize at 60°C, for 3 days. Resin-embedded cells were mounted on stubs using a self-adhesive carbon disk and gold sputtered by an Emithech K550. Regions of interest were cross-sectioned by the focused gallium ion beam of the Dualbeam FIB/SEM (Helios Nanolab, FEI, Hillsboro, OR, USA). Images of cross sections were acquired at a working distance of 2 mm using backscattered electrons and a through-the-lens detector in immersion mode with an operating voltage of 2 kV and an applied current of 0.17 Na. Images were composed in an Adobe Photoshop CS6 format.

After OGD/R and treatment, HBMVECs were fixed using 2.5% glutaraldehyde and then 1% osmium tetroxide (Sigma-Aldrich, USA) for 30 minutes. Dehydration via acetone and embedding via epoxy embedding medium (Sigma-Aldrich, USA) were performed at room temperature. 1 μm ultrathin sections were made and stained by uranyl acetate (Tianfu Chemical Co. Ltd, China). Autophagy was observed by transmission electron microscopy (FEI, Netherlands).

Preparation of ultrathin sections was performed following standard procedure previously published27 (link). In brief: Cleared specimens and unfixed control tissue were postfixed in 1% (wt/v) OsO4 in PBS, and then dehydrated in an ethanol series (50%, 70%, 96%, 100%). The 70% alcohol was saturated with uranyl acetate for contrast enhancement. Dehydration was completed by acetone, followed by propylene oxide. Specimens were infiltrated with rising concentrations of Epoxy embedding medium (Sigma Aldrich, Darmstadt, Germany). Ultrathin sections (60 nm) were cut on a Leica Ultramicrotome (Leica, Bensheim, Germany) and mounted on pioloform-coated copper grids. Ultrathin sections were examined and documented using a LEO 912AB transmission electron microscope (Carl Zeiss, Oberkochen, Germany).

The distribution of SiMAG or fluidMAG in HCECs was investigated using a transmission electron microscope (TEM), as previously described.¹⁹ (link) HCECs were treated with SiMAG or fluidMAG (20 µg/mL) for 24 hours. Cells were fixed overnight with 2.5% glutaraldehyde (Sigma-Aldrich) and 3.7% paraformaldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.6). After washing with 0.1 M phosphate buffer, HCECs were fixed with 1% osmium tetroxide (OsO₄) in the same buffer for 1 hour. Cells were then dehydrated with a graded ethanol series (Merck KGaA, Darmstadt, Germany) before embedding in an epoxy embedding medium (Sigma-Aldrich). Polymerization was then performed at 60°C for 3 days. Ultrathin sections (60–70 nm) of samples were obtained with an ultramicrotome (Leica Ultracut UCT, Leica, Germany). Sections were collected on a grid (200 mesh) and examined using TEM (JEM-1010; JEOL, Tokyo, Japan) at 60 kV. Images were recorded with a charge-coupled device camera (SC1000; Gatan, Warrendale, PA). The length of the electron micrograph was measured with the Gatan Microscopy Suite software (Gatan, Warrendale, PA). Normal control for TEM was incubated in a complete nanoparticle-free medium for 24 hours.