2.7. Microscopic analyses
After 168 hr of incubation at 25 °C, pedicel tissues of ex-vivoexperiments were sectioned vertically, and photographs were taken using Olympus SZX16 microscope under the bright field. For analysis through transmission electron microscopy, AZ containing proximal parts of pedicel tissues were cut into small portions and fixed in Karnovsky’s fixative solution (2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4). Samples were washed with 0.05 M sodium cacodylate buffer and post-fixed for 4 hr in 1 % osmium tetroxide diluted in 0.1 M sodium cacodylate buffer. The samples were then washed with distilled water and stained with 0.5 % uranyl acetate buffer for 16 hr at 4°C. Samples were dehydrated in an increasing ethanol gradient (50 %, 60 %, 70 %, 80 %, 90 %, and 100 %) for 20 min and treated with propylene oxide followed by 1:1, 1:2 propylene oxide: Spurr’s resin solution (10 g of cycloaliphatic epoxide resin (ERL 4221), 6 g of diglycidyl ether of polypropylene glycol (D.E.R. 736), 26 g of nonenyl succinic anhydride, and 0.3 g of dimethylaminoethanol) for 2 hr each. Then, samples were embedded in 100 % Spurr resin solution and sectioned with EM UC7 ultramicrotome (Leica microsystems, Wetzlar, Germany). We observed samples using Carl Zeiss LIBRA 120 transmission electron microscope (Oberkochen, Germany) operating at an acceleration voltage of 120 kV.