Jem 1010 ex electron microscope

Specimens for TEM and SEM were prepared from the maximum tillering stage leaves. For SEM, harvested leaves were fixed overnight at 4°C with slightly modified Karnovsky’s fixative consisting of 2% paraformaldehyde, 2% glutaraldehyde, and 50 mM sodium cacodylate buffer at pH 7.2, and then washed three times with 50 mM sodium cacodylate buffer. Samples were post-fixed with 1% osmium tetroxide in 50 mM sodium cacodylate buffer and then washed three times with distilled water. Samples were treated with 0.5% uranyl acetate, washed with an ethanol gradient series, and then treated with hexamethyldisilazane (HMDS). Samples were mounted on platinum stubs, coated with gold, and examined by a Field-Emission Scanning electron microscope (Sigma, Carl Zeiss).
TEM samples were fixed, post-fixed, and dehydrated as described in SEM, and then embedded in propylene oxide and Spurr’s resin overnight at 70°C. Embedded samples were sliced to 60 mm with an ultramicrotome (MT–X, RMC), and then stained with 2% uranyl acetate for 5 min and Reynold’s lead citrate for 2 min at 25°C. Processed samples were examined using a JEM-1010 EX electron microscope (JEOL, https://www.jeol.co.jp/en/).

Leaf samples for TEM analysis were harvested from 3-month-old plants grown in the greenhouse and from 10-day-old dark-grown etiolated seedlings. Whole tissue preparation was carried out as described previously (Park et al. 2007 (link)). Segments of leaf tissues were fixed in modified Karnovsky’s fixative (2% paraformaldehyde, 2% glutaraldehyde, and 50 mM sodium cacodylate buffer, pH 7.2) and washed three times with 50 mM sodium cacodylate buffer, pH 7.2, at 4 °C for 10 min. The samples were post fixed with 1% osmium tetroxide in 50 mM sodium cacodylate buffer, pH 7.2, at 4 °C for 2 h and briefly washed twice with distilled water at 25 °C. The samples were en bloc stained in 0.5% uranyl acetate at 4 °C for a minimum of 30 min, dehydrated in a gradient series of ethanol and propylene oxide, and embedded in Spurr’s resin. After polymerization at 70 °C for 24 h, the sections were sliced to 60 nm with an ultramicrotome (MT–X; RMC) and stained with 2% uranyl acetate for 5 min and Reynolds’ lead citrate for 2 min at 25 °C. The processed samples were then examined under a JEM-1010 EX electron microscope (JEOL).

Leaf samples for transmission electron microscopy were harvested from five-month-old plants grown under natural long day conditions. Fixation and polymerization of leaf samples were carried out as described previously (Park et al. 2007 (link)). Segments of leaf tissues were fixed in modified Karnovsky’s fixative (2% paraformaldehyde, 2% glutaraldehyde, and 50 mM sodium cacodylate buffer, pH 7.2) and washed three times with 50 mM sodium cacodylate buffer (pH 7.2) at 4 °C for 10 min. The samples were post-fixed with 1% osmium tetroxide in 50 mM sodium cacodylate buffer, pH 7.2, at 4 °C for 2 h and briefly washed twice with distilled water at 25 °C. The samples were then bloc stained in 0.5% uranyl acetate at 4 °C for a minimum of 30 min, dehydrated in a gradient series of ethanol and propylene oxide, and embedded in Spurr’s resin. After polymerization at 70 °C for 24 h, the sections were sliced to 60 nm with an ultramicrotome (MT-X; RMC, http://www.rmcboeckeler.com/) and stained with 2% uranyl acetate for 5 min and Reynold’s lead citrate for 2 min at 25 °C. The processed samples were finally examined using a JEM-1010 EX electron microscope (JEOL, https://www.jeol.co.jp/en/).