2.3.4 Ultramicrotome sectioning
Fixed leaf samples were washed twice in PBS and dehydrated through an
ethanol graded series (30, 50, 70, 90, 100% EtOH). They were then
embedded in medium-grade LR white resin (Agar Scientific), using a
3-step graded series (1:3, 1:1, 3:1 resin:EtOH, 2 h each) and overnight
immersion in 100% resin twice. The resin was polymerized at 60°C for 24
h, semi-thin 2 µm-thick sections were obtained using an ultramicrotome
(EM-UC7, Leica) fitted with a glass blade, and sections were adhered to
glass slides. The sections were stained with 0.5% toluidine blue on a
hotplate for 2 min, washed three times in dH2O, and
mounted in 8:2 glycerol:PBS on a microscope slide. Stained specimens
were imaged with a wide-field microscope (ECLIPSE Ni-U, Nikon) fitted
with a digital camera.