Tecnai 12 transmission electron microscope

Three steps were necessary to obtain the multiscale scaffold (Figure 9).
The topography of the electrospun scaffolds was observed using environmental scanning electron microscopy (Philips XL30 ESEM-FEG). Fiber diameter was measured after setting up the scale bar. Average fiber diameters (n = 100 fibers) were analyzed with ImageJ software (NIH, Bethesda, Maryland). The isotropy value of the scaffold (n = 6) was analyzed using Mountain™ version 8.0 software (with smoothing and maintaining the default frequency thresholds at 5% and 80% Str ISO 25178) and the main orientations of the fibers were analyzed using the Fourier Transformation method (n = 6). Gold deposits over the electrospun scaffold were investigated using Energy Dispersive X-ray Spectroscopy (EDS) analysis with the detector present in the microscope. The measurement is based on the energy and intensity distribution of X-ray signals produced by the electron beam striking the surface of the target scaffold. Samples for transmission electron microscope (TEM) analysis were prepared by dropping a dilute suspension of gold nanoparticles on to copper grids covered by a carbon film. Grids were observed with a Philips Tecnai 12 transmission electron microscope.

For studying cells ultrastructure, the samples were embedded in Spurr resin as described in Garcia de la Garma et al. (2015 (link)). Briefly, samples were fixed for 2.5 h at 4°C in a 0.1 M Na-phosphate buffered (pH 7.2) mixture of 2.5% glutaraldehyde and 4% paraformaldehyde. Tissue was post-fixed with 2% osmium tetroxide for 2 h. The samples were then dehydrated in a graded alcohol series and propylene oxide and embedded in Spurr's resin. Blocks were sectioned on a Leica EM UC6 ultramicrotome, collected on formvar-coated copper grids and stained with uranyl acetate followed by lead citrate. Ultra-thin sections were examined using a Philips Tecnai 12 transmission electron microscope.

Viral morphotypes and purity (free of cell contamination) were determined using transmission electron microscopy. Briefly, one drop of the above purified and concentrated viral solution (approximately 10¹² VLPs/ml) was loaded on a copper grid and dried at room temperature for 15 min. After excess liquid was drained off, the grid was stained with 2% phosphotungstic acid for 2 min. Excess phosphotungstic acid was removed and air-dried. The grids were then examined under a Philips TECNAI 12 transmission electron microscope at an acceleration voltage of 100 kV.

Hippocampal slices were used for electron microscopy analysis according to standard procedures. Ultra-thin sections were examined using a Phillips Tecnai 12 transmission electron microscope at 80 kV at the Electron Microscope Facility of the Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile. For each treatment, 40 to 50 digital images at 16,500X magnification were obtained, and they were manually analyzed with ImageJ in a blinded fashion by measuring the mitochondrial area [32 ,36 (link)]. Ultrastructural features of the mitochondria, such as the integrity of the membrane and cristae, were determined for each mitochondrion concomitantly with the measurement of morphological parameters [37 (link)]. Membranes or cristae were considered to be intact when the entire structure was preserved and organized and the mitochondria appeared normal, as previously described [38 (link)]. Quantitative analysis was performed with n = 3 using GraphPad Prism 5.01 software. For more details, see S1 File.

PEDV CV777 strain was stored in our lab. Protein G-conjugated gold nanorods (120 nm in length and 40 nm in width, ~1.0 × 10¹² particles/mL) were purchased from Creative Diagnostics. (New York, USA). Mouse anti-PEDV polyclonal antibodies were prepared as described in Section 2.2. BSA was purchased from Sigma-Aldrich (St. Louis, MO, USA). PBS was supplied by Biyuntian Co. (Shanghai, China). Tween 20 was received from Aladdin (Los Angeles, CA, USA).
Transmission electron microscopy (TEM) images were obtained using a Tecnai 12 transmission electron microscope (Philips, AMS, The Netherlands). Dark field images were obtained using a Nikon DFM (Nikon, Tokyo, Japan). SEM images were obtained using a GeminiSEM 300 (Carl Zeiss, Oberkochen, Germany) with an acceleration voltage of 10.0 kV and 5 k of magnification. The zeta potential was measured using a Malvern Instrument (Zetasizer NanoES90, Worcs, UK). All fluorescence quantitative data were measured by a LightCycler480 II fluorescence quantitative PCR (qPCR) instrument (Roche, Basel, Switzerland).