Tecnai spirit120kv

Briefly, samples were fixed in 2% PFA and 0.2% GA overnight. After rinsing with phosphate buffer (PB) (0.1 M, pH 7.4), samples were dehydrated through a graded ethanol series (30, 50, 70, 80, 90, and 100%, 10 min each). Samples were infiltrated in a graded mixtures (3:1, 1:1, 1:3) of ethanol and LR White resin (Ted Pella) then changed two to three times pure resin. Finally, samples were embedded in pure resin and polymerized for 48 h at 50°C. The ultrathin sections (70 nm thick) were sectioned with microtome (Leica EM UC6). After rinsing with 0.1 M PB, sections were blocked in 5% goat serum in 1% BSA buffer (with 0.15% Glycine in PB) for 30 min, then incubated with 1:10 diluted Rabbit Anti-mCherry antibody (ab167453, Abcam, Cambridge, United Kingdom) for 2 h. After rinsing with 0.1 M PB six times again, sections were followed by immunogold labeled with 10-nm protein A-gold (1: 50; Cell Microscopy Center, University Medical Center Utrecht, Utrecht, Netherlands) for 1 h. Following rinses with PB, sections were stained with 2% uranyl acetate for 5 min, and imaged by a transmission electron microscope (FEI Tecnai Spirit 120kV).

For transmission electron microscopy (TEM), control and Tet DKO BMMSCs were collected by centrifugation and fixed with 2.5% glutaraldehyde overnight and then fixed with 1% osmium tetraoxide for 2 h, dehydrated in a graded series of ethanol concentrations, and embedded in SPIPON812 resin and polymerized. The block was sectioned by microtome (Leica EM UC6). The ultrathin sections approximately 70 nm, mounted on copper grids, uranyl acetate, and lead citrate were used to stain, and examined, and photographed with a FEI Tecnai spirit TEM (FEI Tecnai Spirit 120 kv).

For TEM analysis, cells were fixed with 2.5% (v/v) glutaraldehyde with phosphate buffer (0.1 m, pH 7.4), washed four times in phosphate buffer at 4 °C. Cells were postfixed with 1% (w/v) osmium tetroxide (OsO₄) and 1.5% (w/v) potassium ferricyanide aqueous solution at 4 °C for 2 h, dehydrated through a graded ethanol series (30, 50, 70, 80, 90, 100, and 100%, 5 min each at 4 °C) into pure acetone (2 × 5 min). Samples were infiltrated in a graded mixture (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (16.2 ml of SPI-PON812, 10 ml of dodecenyl succinic anhydride and 8.9 ml of methyl nadic anhydride, then changed to pure resin. Finally, cells were embedded in pure resin with 1.5% N,N-Dimethylbenzylamine and polymerized for 12 h at 45 °C, 48 h at 60 °C. The ultrathin sections (70 nm thick) were sectioned with microtome (Leica EM UC7), double-stained by uranyl acetate and lead citrate, and examined by a transmission electron microscope (FEI Tecnai Spirit120kV).

For ultrastructural analyses, infected HFF cells were fixed with 1% glutaraldehyde (Polysciences, Inc.) and 1% osmium tetroxide (Polysciences, Inc.) in 50 mM phosphate buffer at 4°C for 30 min. The samples were then processed as described previously (19 (link)). Ultrathin sections (70 nm thick) were prepared with a microtome (Leica EM UC6), double-stained with uranyl acetate and lead citrate, and examined with a transmission electron microscope (FEI Tecnai Spirit120kV).

RD cells grown on 10-cm dishes were mock-infected or infected with EV71 (MOI = 10) for 12 h. The cells were harvested and washed three times with PBS and then frozen using a high-pressure freezer (Leica EM HPM100). Freeze substitutions were performed in a substitution unit (Leica EM AFS2) in dry acetone with 2% osmium tetroxide at −90°C for 72 h followed by gradual warming to −20°C for 8 h and 0°C for 2 h. After washing with dry acetone at 0°C three times, the samples were warmed to room temperature. After infiltration with SPI812 resin for several hours, the samples were embedded into SPI812 resin, placed in a 60°C oven for 24 h, and then sectioned using a microtome (Leica EM UC6). Ultrathin sections were stained with uranyl acetate and lead citrate and then examined by transmission electron microscopy (FEI Tecnai Spirit 120 KV).

The two longest tracheal tubes linking the largest spiracle on both sides of the thorax and the spiracle basal to head of the beetle were dissected and instantly fixed with 2.5% (vol/vol) glutaraldehyde (Coolaber, SL1790) and 4% (vol/vol) paraformaldehyde (Solarbio, P1112) with phosphate buffer (PB) (0.1 M, pH 7.4) (Macklin, H885798). The samples were then washed four times in PB and then tubes were postfixed with 1% (wt/vol) osmium tetraoxide in PB for 2 hr at 4°C, dehydrated through a graded ethanol series (30, 50, 70, 80, 90, 100%, 100%, 7 min each) into pure acetone (2×10 min). Samples were infiltrated in graded mixtures (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (16.2 g SPI-PON812, 10 g DDSA, and 8.9 g NMA), and then changed to pure resin. Finally, tubes were embedded in pure resin with 1.5% BDMA and polymerized for 12 hr at 45°C, 48 hr at 60°C. The ultrathin sections (70 nm thick) were sectioned with microtome (Leica EM UC6, Germany), double-stained by uranyl acetate and lead citrate, and examined by a transmission electron microscope (FEI Tecnai Spirit120kV, OR).

For TEM analysis, MNT1 cells transfected with siRNAs for 48 h were fixed with 2.5% (vol/vol) glutaraldehyde with phosphate buffer (0.1 m, pH 7.4) and washed four times with phosphate buffer at 4°C. For the eyes of mice, samples were rinsed with 0.1 M phosphate buffer saline (pH 7.2) and placed in 2.5% glutaraldehyde for 8 min. The cornea was cut, the lens was extruded, and then the samples were kept in 2.5% glutaraldehyde overnight. Samples were postfixed with 1% (wt/vol) osmium tetroxide (OsO₄) and 1.5% (wt/vol) potassium ferricyanide aqueous solution at 4°C for 2 h. Samples were dehydrated through a graded ethanol series (30, 50, 70, 80, 90, 100, and 100%, 5 min each at 4°C) into pure acetone (2 × 5 min), infiltrated in a graded mixture (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (16.2 ml of SPI-PON812, 10 ml of dodecanoyl succinic anhydride and 8.9 ml of methyl nadic anhydride), and then changed to pure resin. Finally, samples were embedded in pure resin with 1.5% N, N-dimethylbenzylamine and polymerized for 12 h at 45°C and 48 h at 60°C. The ultrathin sections (70-nm thick) were prepared with a microtome (EM UC7; Leica), double-stained by uranyl acetate and lead citrate, and imaged by a TEM (FEI Tecnai Spirit 120 kV).