Ultracut uct

Specimens for electron microscopy were immediately fixed in 2.5% phosphate-buffered glutaraldehyde (pH 7.4), post fixed in 1% osmium tetroxide in the same buffer at 4 °C and dehydrated. For transmission electron microscopy, the specimens were embedded in epoxy resin. Ultrathin sections (60–70 nm) were obtained (Leica ultra-cut UCT) and stained with uranyl acetate and lead citrate⁷² , examined and photographed (JEOL JEM 2100 electron microscope; Jeol Ltd, Tokyo, Japan) in Electron Microscope Research Laboratory (EMRL) of Faculty of Agriculture, El Mansoura University, Egypt.

Toluidine blue stain

Semi-thin sections (1 µm) were also obtained (Leica ultra-cut UCT), stained with toluidine blue and examined by light microscope.

Cells were fixed with 2% glutaraldehyde for 5 hrs and postfixed with 0.5% paraformaldehyde; washed with a 0.1 M cacodylate buffer (pH = 7.4), contrasted with a 1% OsO4 solution in a cacodylate buffer (pH = 7.4), dehydrated in an increasing series of ethanol solutions, followed by dehydration with acetone, impregnated, and embedded in Epon-812 (following manufacturer’s instructions). Ultrathin sections (100–200 nm thick) were cut with a diamond knife (Diatome) on an ultramicrotome Ultracut-UCT (Leica Microsystems), transferred to copper 200 mesh grids, covered with formvar (SPI, Washington, DC, USA), and contrasted with lead citrate, according to the Reynolds established procedure [46 (link)]. For the analytical electron microscopy study, contrasting was omitted in some cases.
Ultra-thin sections were examined in transmission electron microscopes JEM-1011 and JEM-2100 (Jeol, Tokyo, Japan) with accelerating voltages of 80 kV and 200 kV, respectively, and magnification of ×13,000–21,000. Images were recorded with Ultrascan 1000XP and Orius SC1000D CCD cameras (Gatan, Pleasanton, CA, USA).

P1 pups were anesthetized and perfused with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. 1-mm³ dorsal skin section was taken from the perfused pups and placed in the same fixative for an additional 2 h at 4 °C. The skin samples were rinsed three times in 0.2 M sucrose/ sodium cacodylate buffer and were left in this buffer overnight. The samples were then post-fixed in ferrocyanide-reduced osmium tetroxide for 1 h at 4 °C followed by dehydration with acetone, infiltration with acetone/epon, and embedded in Epon. Sections were cut with Leica Microsystems Ultracut UCT. To examine the sections, FEI Tecnai 12 BioTwin 120 kV Transmission Electron Microscope imaged with an AMT XR80C CCD Camera System.