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.