Spurr s epoxy resin

Mesenchymal stromal cells in culture were fixed in 4% formaldehyde and 4% glutaraldehyde (catalog # 16000; Electron Microscopy Sciences, Hatfield, PA, USA) in PBS overnight, post-fixed in 2% osmium tetroxide (catalog # 19150; Electron Microscopy Sciences) in PBS, dehydrated in a graduated series of ethanol solutions, and embedded in Spurr's epoxy resin (catalog # 14300; Electron Microscopy Sciences). Blocks were processed as described previously 6 . The sections of embedded specimens were analysed with a Philips CM100 electron microscope.

For ultrastructural collagen analysis, normal skin and wound tissues were fixed in 4% glutaraldehyde. After rinsing with 0.1 mol/L sodium cacodylate buffer, samples were post-fixed in 1% osmium tetraxide, dehydrated in graded alcohols, and embedded in Spurr's epoxy resin (Electron Microscopy Sciences, Fort Washington, PA). Ultrathin sections (80 nm) were collected on grids, stained with uracyl acetate and lead citrate and examined using a Hitachi H-600 (75 kV) TEM as previously described [26] (link). The wound bed was identified using a serial section stained with toluidine blue. TEM images of collagen fibril cross-sections were taken at random within the boundaries of the wound bed at a magnification of 30,000× and the photographic negatives were scanned into Adobe Photoshop. The diameter of the collagen fibrils was measured using Scion Image. Five normal skin samples and five wound samples from each time point, all from different mice, were used for analysis. Each electron micrograph was divided into four parts for analysis, and fibrils from four micrographs per sample were measured. A total of 1100–2100 fibrils per animal were analyzed [26] (link). Images of fibroblasts within the wound bed were captured at 8000× and the photographs were scanned into Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA).

Cells were grown in six‐well plates, three control wells were treated with oleic acid for 24 h and three treatment wells were treated with oleic acid for 24 h plus 0.5 mM caffeine for 6 h. Cells were harvested and fixed in a modified Karnovsky's fixative (2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M Sorenson's phosphate buffer, pH 7.4) overnight at room temperature. Cells were rinsed in Sorenson's phosphate buffer, followed by secondary fixation in 1.0% osmium tetroxide with 0.8% potassium ferricyanide in 0.2 M cacodylate buffer, pH 7.4. Samples were rinsed and dehydrated with an ethanol series, and embedded in Spurr's epoxy resin (Electron Microscopy Sciences). Embedded samples were trimmed, and 90 nm sections were cut on a Leica UC7 ultramicrotome (Leica Microsystems, Inc.). Sections were collected on Formvar‐coated (0.25 g Formvar in 100 ml ethylene dichloride), 200 mesh copper grids. Sections were stained with 2% uranyl acetate in 50% ethyl alcohol for 15 min, rinsed with DI, and stained with Reynolds lead citrate for 15 min. Sections were imaged on a Tecnai 12 Spirit TEM (FEI). A total of 30 images were collected for each treatment group and images were analyzed in Fiji (Schindelin et al., 2012). Point counting stereological analysis was used to determine volume density of autophagosomes and mitochondrial.

Immediately after harvesting, roots of ten DAG seedlings were fixed overnight in ice-cold 0.1 M cacodylate buffer, pH 7.2, containing 2.5% (v/v) glutaraldehyde. They were then washed in the same buffer, post-fixed in 1% OsO₄ for 2 h at room temperature (RT) and washed again in cacodylate buffer, pH 7.2. After dehydration in a graded ethanol series samples were embedded in Spurr’s epoxy resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections (70 nm thick) were cut on an RMC ultramicrotome (RMC, Tucson, AZ, USA) and mounted on 150 mesh formvar-coated copper grids (Electron Microscopy Sciences). Just before analysis, the sections were stained with uranyl acetate for 30 min at room temperature, washed with ultrapure water and stained 10 min with lead citrate and washed again with ultrapure water. Images were taken with a JEOL100 CXII instrument (JEOL USA Inc., Peabody, MA) equipped with Gatan SC1000 (Model 832) CCD camera (Gatan Inc., Pleasanton, CA, USA).