Veleta ccd digital camera

Cardiac samples from animals of the different groups (n = 3/group) were fixed with 1% Glutaraldehyde (8% Aqueous Solution, Electron Microscopy Sciences) in Sodium Cacodylate buffer 0.2 M pH 7.4. The specimens were washed in distilled water, stained with 2% Osmium tetroxide and 0.5% Uranyl acetate, dehydrated through a graded series of ethanol ethanol (50, 70, 90, and 100%), Propylene Oxide and embedded in Epon resin (Sigma) and polymerized at 60°C for 72 h. Ultrathin (60 nm) sections were cut with a Leica EM UC7 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and mounted on grids. Images were acquired from thin sections using a FEI Tecnai 12 transmission electron microscope (FEI Company, Hillsboro, Oregon, USA) equipped with a Veleta CCD digital camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Count and quantification of mitochondria was performed using the iTEM software (Soft Imaging Systems GmbH, Münster, Germany). For quantification, 15 random regions for each sample were considered.

Negative stain treatment covers the sample with a layer of heavy metal salts so this contrast allows visualization of the sample surface. 5 μL  of S-EVs were resuspended in PBS containing 4% Paraformaldehyde (1:1) to fix them. The sample was applied to formvar 100-mesh grids and incubated for 10 min. Grids were washed twice with filtered distilled water and stained using 1.5% UA in water for 10 min. After they were washed with water to remove the excess staining solution and grids were air-dried. Images were acquired from grids using a FEI Tecnai 12 transmission electron microscope (FEI Company, Hillsboro, Oregon, USA) equipped with a Veleta CCD digital camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany) and operating at 120 kV. Images were collected at magnifications of 21000X, 30000X and 68000X.

Cardiac sample sections from different treatment groups were cut into 1-mm-thick slices and fixed with glutaraldehyde in sodium cacodylate buffer overnight at 37°C. The specimens were washed in distilled water, stained with 2% osmium tetroxide and 0.5% uranyl acetate, dehydrated through a graded series of ethanol and propylene oxide, embedded in epoxy resin, and polymerized for 3 days. Ultrathin sections of 90 nm were cut and mounted on Formvar-coated slot copper grids. Images were acquired of the thin sections using a FEI Tecnai 12 transmission electron microscope (Hitachi H-600, Tokyo, Japan) equipped with a Veleta CCD digital camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). For quantification, 20 random regions for each sample were considered.