Jem 1400 transmission electron microscopy

Lung tissues were fixed in fresh 3% glutaraldehyde for at least 4 hrs at 4°C, post-fixed in 1% osmium tetroxide for 1.5 hrs, dehydrated in a gradient series of ethanol, infiltrated with Epon 812, embedded and cultured at 37, 45 and 60°C for 24 hrs. Ultrathin sections were ultracut using an ultracut E ultramicrotome and stained with uranyl acetate and lead citrate prior to observation under a JEM-1400 transmission electron microscopy (TEM; Jeol Ltd).

The brains were harvested two days after surgery. As described previously [9 (link)], sections of the hippocampal CA1 region were prepared for electron microscopy by fixation in 0.5% glutaraldehyde and 1% osmium tetroxide. Samples were dehydrated in acetone solutions at increasing concentrations, embedded in an epoxy resin, and stained with uranyl acetate and lead citrate. The ultrastructural changes of the basal lamina, tight junction and mitochondria, as well as the angioedema surrounding the capillaries all indicative of BBB integrity disruption were observed with JEM-1400 transmission electron microscopy (JEOL, Tokyo, Japan).

For cellular ultrastructural analysis, glands at 72 h of culture were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer and kept in the fridge until processing [43 (link)]. Tissues were then rinsed in 0.1 M phosphate buffer three times. Post-fixation was done in 2% osmium tetroxide in the same buffer solution at 4 °C for 45 min, and tissues were dehydrated in a graded series of alcohol and embedded in Spurr’s resin:propylene oxide (1:1) for 10 min, Spurr’s resin:propylene oxide (3:1) for 15 min, and 100% Spurr’s resin for 15 min three times. The embedding process continued for 16 h at 70 °C. Semi-thin sections were obtained using glass knives with Ultracut E Microtome (Leica Microsystems, Wetzlar, Germany), and ultra-fine sections (90–100 nm) were mounted on copper grids of 100 meshes. The grids were stained by uranyl acetate and lead then observed in a JEM-1400 transmission electron microscopy (JEOL, Peabody, MA, USA) adjusted to 200 kV. Control glands (non-irradiated) were used as positive controls. According to random regions of interest in electron micrographs from end bud regions containing ten epithelial cells. Apoptotic bodies were counted using ImageJ software.