Spurr

Cells were grown to 90% confluence before being washed, scraped, pelleted, and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) overnight. Samples were post fixed in 1% osmium tetroxide for 2 h, stained in bloc with 0.5% uranyl acetate, rinsed, and dehydrated in graded ethanol. After immersion in propylene oxide, samples were embedded in epoxy resin (Spurr, Electron Microscopy Sciences, EMS, Hatfield PA, USA) and polymerized for 42 h at 75 °C. Ultrathin sections were stained with lead citrate and uranyl acetate and examined in a FEI Tecnai G20 Electron microscope at 200 kVA.

Cells were grown to 90% confluence before being washed and cultured in serum-free medium for 24 hours. Cells were then scraped off, pelleted, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) overnight, and postfixed in 1% osmium tetroxide in the same buffer for 2 hours. Samples were then washed in distilled water, stained in bloc with 0.5% uranyl acetate, rinsed and dehydrated in graded ethanol. After immersion in propylene oxide, samples were embedded in epoxy resin (Spurr, Electron Microscopy Sciences, EMS, Hatfield PA, USA) and polymerized for 42 hours at 75°C. Sections (0.5 μm-thick) were stained with 1.0% toluidine blue in 1.0% aqueous sodium borate for light microscopic examination. Ultrathin sections were stained with lead citrate and uranyl acetate and examined in a JEOL 1010 transmission electron microscope (Jeol Inc., Peabody, MA, USA) at 80 kV.
Extracellular vesicles were obtained and pelleted by centrifugation as described above, suspended in PBS, and 5 μl of samples were deposited on carbon coated copper grids (CF100-Cu, EMS), incubated at room temperature overnight, and fixed with 2.5% glutaraldehyde for 5 minutes. Grids were washed with distilled water several times and stained with 1% uranyl acetate for 5 minutes, followed by TEM analysis.

For conventional transmission electron microscopy (TEM), vitellogenic oocytes were fixed in freshly prepared 4% formaldehyde, 2.5% glutaraldehyde diluted in 0.1 M sodium cacodylate buffer pH 7.3 (Electron Microscopy Sciences) at 4^ºC for 24 h, and then embedded in epoxy resin (Spurr) (Electron Microscopy Sciences), sectioned and stained using standard methods. For freeze fracture, the yolk organelles were obtained and processed as described in [35 (link)]. All samples were examined in a JEOL 1200 EX transmission electron microscope, operating at 80 kV.

Skin transversal samples from the equatorial regions of the three cashew pseudofruits were fixed in Karnovsky’s solution with modifications (2.5% glutaraldehyde, 2.5% paraformaldehyde, and 0.05 mM CaCl₂ in sodium cacodylate buffer (0.1 M, pH 7.2)) [46 (link)] for 48 h. The samples were post-fixed in 1% osmium tetroxide for 2 h, dehydrated in increasing acetone series up to 100%, and infiltrated in resin (Spurr, Electron Microscopy Sciences, Hat Field, PA, USA). The blocks were sectioned in the ultramicrotome (UC6, Leica, Vienna, Austria). Sections were mounted on 200-mesh copper screens and contrasted with 5% uranyl acetate and 2% lead citrate for 30 min in each step [50 (link)]. Observations and electron micrographs of the epidermis and cuticle were performed using a transmission electron microscope (JEM 1011, JEOL, Akishima, Japan) with a coupled Gatan 830 J46W44 video camera operating at 60 Kv.