Tecnai 10

Replicas were imaged with a Tecnai 10 (FEI, 80kV accelerating voltage) or Tecnai 12 electron microscope (FEI, 120kV accelerating voltage). Images of E-face PSDs of spine synapses and P-face AZs putatively contacting spine synapse, identified by the shallow concave AZ structure, were taken from middle third of SR and from SO of the same replica at a similar medial-lateral position. The sample was tilted in order to maximize the synaptic membrane area on the image. Synapses containing at least 2 gold particles (irrespective of size) were included in the analysis, except when quantifying the percentage of synapses expressing AMPAR, where all morphologically identified PSDs were included in the analysis. When demarcating manually, we defined the PSD as area of tightly packed IMP clusters (distance between IMPs ≤ 15 nm). AZ was demarcated based on alteration in surface curvature and/or higher density of IMPs following previously reported practice [44 (link)]. Gold particles were counted if their center was inside or ≤ 30 nm away from the demarcation border.

The ultrastructure of endometrial tissues was observed with transmission electron microscopy (FEI Tecnai T10, USA, Center of Cryo-Electron Microscopy, Zhejiang University). The samples were cut into 1 × 1 × 1 mm³ cubes and fixed in 2.5% glutaraldehyde at 4 °C overnight. Then the samples were postfixed with 1% osmic acid at ambient temperature for 1 h and stained with uranyl acetate for 0.5 h. After dehydration with gradient ethanol and 100% acetone, samples were permeabilized for 2 h each with a 1:1 and 3:1 embedding agent/acetone at room temperature and then placed in pure embedding agent, polymerized, and sectioned with a microtome (Leica EM UC7, Vienna, Austria). Images were collected by a Tecnai 10 (FEI, 120 kV accelerating voltage) electron microscope. We observed the number and length of mitochondria, and the width of cristae junction and cristae in three groups by Image J software (https://imagej.nih.gov/ij/).

ST possesses bristle‐like type 1 fimbriae which are 5 – 7 nm in diameter and about 1 µm long and flexible flagella which are about 20 nm in diameter and 15 – 20 µm long (Althouse et al., 2003). Bacteria cultured 16 h in the same concentration of compound used in the invasion assays (the NIC, Table 1) were prepared for electron microscopy as follows. The coated side of a copper grid, coated with 0.7% Formvar (Agar Scientific, Essex, UK), was incubated on a drop of bacterial culture for 4 min, allowed to dry in air, rinsed twice with distilled water, dried, stained 3 min with 2% uranyl acetate solution and dried again. Samples were observed under a FEI Tecnai 10 transmission electron microscope with a SIS (Olympus) Megaview II side entry 1 K camera and compared to untreated control cultures.

Five rats that undertaken the same procedure were used for Lanthanum nitrate tracing under TEM. After aortic infusion for 5 min with heparinized saline, 1 mm³ of the brain samples at the same part of two hemispheres was fixed at 4°C overnight with 0.1% glutaraldehyde, 2% Lanthanum nitrate, 2% polyformaldehyde, and 3% sodium cacodylate perfusion. Specimen of the brain tissue was washed twice with Lanthanum-containing cacodylate buffer, fixed with Lanthanum-containing 1% osmium tetroxide. After acetone gradient dehydration and Epon 812 embedding, thin slices were cut with microtome (LKB4800A, Microm, Germany) and observed under electron microscopy (TECNAI-10, FEI, Netherland).

Of two animals of each group, the left lung was cut out with the bronchus attached and fixed by pressure (2 kPa for 1 hour) with half-strength Karnovsky fixative (0.08 M Sodium-cacodylate buffer, 2.5% glutaraldehyde, 0.025 mM CaCl₂, 0.05 mM MgCl₂) for electron microscopy. After two days, the fixative was replaced with 2% para-formaldehyde in sodium-cacodylate buffer for storage. Of each lung, three slices of about 1 – 1.5 mm were cut out at the top, middle and bottom of the lung for further analysis. The tissue slices were post-fixed by osmium potassium ferro-cyanide solution (0.1 M sodium-cacodylate buffer, 1% osmium tetra-oxide, 1.5% potassium ferro-cyanide). Next, the tissue slices were dehydrated in aceton series, cut in smaller pieces and subsequently embedded in epon. The epon was polymerized at 60°C. The epon blocks were trimmed prior to sectioning. During sectioning, ultrathin sections of 70 nm were sectioned and put on 3 mm hexagonal copper grids coated with a formvarfilm. The sections were analysed by transmission electron microscopy (TEM) (Tecnai 10, 100 kV, FEI, Eindhoven, the Netherlands).

Characterizations of the particles were performed by transmission-electron microscopy (TEM) using freeze-fracture preparation technique and by static light scattering (SLS). Sonicated and autoclaved stock solutions of NPs were diluted 1:10 in cell culture medium and cryoprotected by immersion in glycerol solution. Samples were cryofixed into melting Freon 22 and liquid nitrogen. Freeze fracturing took place at −120 °C with a BAF 400 (Bal-Tec, Balzers, Liechtenstein). Freeze-fractured specimens were replicated by application of Pt/C and C by electron-gun evaporation. The replicas were cleaned in concentrated sodium hypochlorite and in acetone and examined with a Tecnai 10 (FEI Company, Hillsboro, OR, USA) transmission-electron microscope operated at 80 kV. Size measurement of single particles and particle aggregations using the TEM images was conducted with the program “Bild-Vermessen 1.0” (CAD-KAS Kassler Computersoftware GbR, Markranstädt, Germany). At least 30 single particles and 20 aggregates of the different NPs were measured. Particle size distribution was measured by SLS using a LS 230 (Beckmann Coulter, Krefeld, Germany). The particle dispersion was dosed into the instrument without special sample preparation. The volume fraction-length mean diameter was measured.