Jem 1010 tem

For cell lines, ∼5,000 cells were plated in each well of 12-well plates and treated with the drug for 6 days. For xenografts, tumours were harvested and samples were cut into 1-mm³ pieces. The cell lines and tumour samples were processed similarly and Electron microscopy was performed at the High Resolution electron microscopy facility at MD Anderson Cancer Center as described previously65 (link). Briefly, they were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3. Samples were then washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid. They were fixed with 1% buffered osmium tetroxide for 30 min and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, filtrated and embedded in LX-112 medium. They were then polymerized in a 60 °C oven for approximately 3 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 TEM (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 80 kV. Digital images were obtained at magnifications of × 5,000, × 25,000 and × 50,000 using the AMT Imaging System (Advanced Microscopy Techniques Corp, Danvers, MA).

For fluorescence microscopy, a fluorescence microscope NIKON ECLIPSE TE2000-U, connected to a high resolution HAMAMATSU ORCA-ER camera was used. For SEM, used to visualize protoplast cell wall regeneration, cells were fixed with 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer containing 1 M sorbitol at 4°C, overnight. Post-fixation was carried out for 2 h at room temperature with 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2). Initial dehydration was accomplished in a graded ethanol series. Then, samples were dehydrated with acetone until dried by the critical point method in liquid CO₂ (Balzers^® CPD 030). Subsequently, the specimens of the different strains were coated with graphite and gold in a vacuum evaporator (EMITECH SCD 004) and examined with a SEM JEOL Observations were carried out in JEOL JSM-6400 microscope SEM. For transmission electron microscopy (TEM), used to observe cell wall, cells were fixed in 4% paraformaldehyde, 1%, glutaraldehyde, and 0.1% PBS overnight at 4°C. Samples were incubated for 90 min in 2% osmium tetroxide and then serially dehydrated in ethanol and embebbed in EMBed-812 resin (Electron Microscopy Sciences). Thin sections (50–70 nm) were obtained by ultracut and observed in JEOL JEM 1010 TEM.

Endothelium-Descemet’s tissue samples from WT and KO mice were fixed with 2.5% glutaraldehyde (#16020, Electron Microscopy Sciences, Hatfield, PA, United States), 4% paraformaldehyde (#15710, Electron Microscopy Sciences), in 0.1 M sodium cacodylate buffer, pH 7.2 at 4°C and post-fixed with 1% osmium tetroxide (# 19,150, Electron Microscopy Sciences) in 0.1 M sodium cacodylate buffer (#12300, Electron Microscopy Sciences), pH 7.2 at 4°C. Samples were dehydrated in a graded ethanol series to 100% ethanol, transitioned to propylene oxide (#20401, Electron Microscopy Sciences), and infiltrated with Embed 812 resin (#14120, Electron Microscopy Sciences). Infiltrated samples were placed in flat embedding molds and polymerized at 65°C for 18 h. Resin blocks were cut with a diamond knife using a Leica Ultracut UCT ultramicrotome (Leica Systems, Buffalo Grove, IL). Sections were placed on 300 mesh copper TEM grids (#0300-CU, Electron Microscopy Sciences) and stained with saturated uranyl acetate in aqueous solution, and lead citrate. Stained sections were viewed with a JEM-1010 TEM (JEOL, Peabody, MA, United States) at 80 kV and photographed with a Gatan MegaScan 794 CCD camera or JEM-1400plus TEM (JEOL) with a Gatan OneView CMOS digital camera.

Samples from each COMECS (Control and FFSF) were examined by transmission electron microscopy (TEM). Samples were immersed in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS). After glutaraldehyde fixation they were given three 15 minute washes in PBS. Following this they were placed in 2% aqueous osmium tetroxide for 2 hours, after which they were given three 15 minute washes in PBS. Following this the samples were dehydrated through a series of ethanol solutions (50, 70, 80, 90, 95 and 100%). For TEM the samples were embedded in araldite resin (Agar Scientific Ltd., Stansted, UK); sections 70 nm thick were cut with a diamond knife and collected onto 300 mesh copper grids. The grids were stained for 1 hour with uranyl acetate, washed in distilled water then stained for 30 minutes with 1% phosphotungstic acid, washed with distilled water and then stained for 20 minutes with Reynolds’ lead citrate. The grids were then examined in a JEM 1010 TEM (JEOL Ltd., Tokyo, Japan).

Monolayers of BHK-21 cells grown on tissue culture plates were infected with FMDV (MOI 100). After 1 h adsorption at 4 °C, the samples were shifted to 37 °C for 5 min. Cell were washed three times with PBS, and cell scarpers were used for the mechanical harvesting of cells. Cell cultures were collected in 1.5 ml Eppendorf tubes and centrifuged at 1000rpm for 5min. The cells pellets were fixed in 2.5% glutaraldehyde at room temperature for 4 h, washed three times in PBS, postfixed with 1% osmium tetroxide for 1 h at 4 °C and washed three times in PBS. Then, samples were dehydrated with gradient ethanol from 50 to 100% (concentrations used were 50, 70, 80, 90, 95 and 100%) for 20min every time and then embedded in epoxy resin. Ultrathin sections were then obtained using an ultramicrotome (Reichert-Jung, Heidelberg, Germany) and double stained by uranyl acetate and lead citrate for 15min at room temperature, respectively. Finally, the sections were examined at 120 keV under a JEM-1010 TEM (JEOL, Tokyo, Japan).

Microscopy analyses were performed at the CACTI, University of Vigo. A JEOL JEM1010 TEM working at 100 kV was used to obtain low-magnification TEM images. A JEOL JEM 2010F field-emission gun TEM working at 200 kV was used to obtain HRTEM images. EDS maps were acquired by coupling the scanning unit of the microscope to an INCA 200 EDS system. EEL spectra were collected in STEM mode using a Gatan GIF Quantum spectrometer with an energy resolution of 1.75 eV (FWHM Zero Loss peak), 0.5 eV/channel energy dispersion and an EELS collection semi-angle of 16 mrad. The EEL spectral background was subtracted using standard DigitalMicrograph routines. All TEM samples were prepared by placing drop of the sample on a TEM copper grid coated with holey carbon thin film and then air dried. To avoid the interference due to the carbon foil grid, the EEL spectra were collected from areas of sample situated in a hole.