Jfd 300

For standard SEM observation, samples were prefixed with 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and postfixed in 1% OsO₄ in the same buffer. The specimens were then dehydrated, freeze dried (JFD300, JEOL) and ultra-thin coated with OsO₄ (PMC-5000, Meiwa). For TEM to observe the surface fine structure of the samples, specimens were prefixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), and then postfixed in 1% OsO₄ in the same buffer. The dehydrated specimens were embedded in an Epon–Araldite mixture. Ultra-thin sections (approximately 70 nm) were cut vertical to the surface of the sample. Sections were stained with 2% uranyl acetate followed by 0.4% lead citrate for 5 min each. To make the SSE-based NanoSuit visible, 10% platinum blue (Nisshin EM) was added to the SSE solution used to treat samples (figure 3).

For conventional SEM observations, petals of the flowering cherry were prefixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH7.4) and postfixed in 1% OsO₄ in the same buffer. The specimens were then dehydrated, freeze dried (JFD300, JEOL), and ultra-thin coated with OsO₄ (PMC-5000, Meiwa). For transmission electron microscopy (TEM), specimens were prefixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH7.4), and then postfixed in 1% OsO₄ in the same buffer. The dehydrated specimens were embedded in an Epon-Araldite mixture. Ultra-thin sections (approximately 70 nm) were cut (ULTRACAT OmU₄, REICHERT-JUNG) vertical to the surface and were stained with 2% uranyl acetate followed by 0.4% lead citrate for 5 min each.

The fixed oviducal glands with or without tendrils were rinsed in 70% ethanol. Micrographs of oviducal gland and oviduct were obtained under a stereomicroscope (M165FC; Leica, Wetzlar, Germany) attached to a digital camera (Wraycam-noa630; Wreymer, Osaka, Japan). Diameter of tendril was determined using a digital microscope VHX-7000 and a software package (Keyence, Osaka, Japan).
For scanning electron microscopy observation, the tendril-forming area of the oviducal gland was trimmed to an approximately 1-cm cube, dehydrated in graded concentrations of ethanol, immersed in butyl alcohol, and dried in a vacuum freeze-dryer (JFD-300; JEOL Ltd, Tokyo, Japan) overnight. Tissues mounted on a sample holder were coated with platinum palladium using an ion sputtering apparatus (E-1030; Hitachi High-Technologies Corp., Tokyo, Japan) and were observed using a scanning electron microscope (s-4800; Hitachi High-Technologies Corp.).

Melon roots were pre-inoculated with conidial suspension (1 × 10⁸ conidia/mL) of the GFP-expressing strains by the root-dip method and planted in the soil infested with Mel02010-DsRed (1 × 10⁵ conidia/g soil). Three plants were removed from the soil at 3 and 7 days after planting, and conidial germination and hyphal elongation on main root surface were observed using a confocal laser scanning microscope (CLSM) (LSM-700; Carl Zeiss, Oberkochen, Germany) (GFP: excitation 488 nm and emission 509 nm; DsRed: excitation 555 nm and emission 572 nm). At least six sections of the main roots of each seedling were observed, and germination rates of conidia and hyphal lengths were measured. Data were analyzed for significant differences using Tukey–Kramer’s multiple range test.
For observing hyphae on the root surface using a scanning electron microscope, sections of the main roots were fixed twice in 2% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h and dehydrated using a graded ethanol series (20–100%), then immersed in 100% acetone. Samples were freeze-dried (JFD-300; JEOL, Tokyo, Japan), coated with a thin gold layer using a JEE-400 vacuum evaporator (JEOL) and observed using a JSM-5800 scanning electron microscope (JEOL).

For transmission and scanning electron microscopy, the embryos at E9.25-E9.75 were fixed in 2.5% glutaraldehyde dissolved in 0.1 M phosphate buffer (pH 7.4) for at least 24 h at 4°C, and then postfixed using 1% osmium tetroxide (OsO₄) dissolved in 0.1 M phosphate buffer (pH 7.3) for 1 h. For transmission electron microscopy, the embryos were postfixed with 0.5% OsO₄ suspended in 0.1 M phosphate buffer (pH 7.3) for 30 min and dehydrated in a graded series of ethanol concentrations. After passage through propylene oxide, the tissues were embedded in Epon 812. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and then observed with a transmission electron microscope (JEOL, Tokyo, Japan, JEM-1010C). For scanning electron microscopy, the specimens were dehydrated in a graded series of ethanols (50%, 70%, 90%, 99.5%, and 100%), critical-point dried with carbon dioxide using a freeze-drying device (JEOL JFD-300), mounted and then coated with gold using a sputter coater. Finally, the specimens were observed under a scanning electron microscope (JEOL JSM-5600 LV SEM).

Cells were fixed and collected by the same methods used for determination of cell densities. Cells on a filter were then dehydrated through a graded ethanol series, substituted with t-butyl alcohol, and finally freeze-dried (JFD-300, JEOL, Tokyo, Japan [22] (link)). The dried cells coated with gold-palladium using an ion sputter (JFC-1500, JEOL). Samples were observed under SEM (SU1510, Hitachi High-Technologies Co., Tokyo, Japan).
For the quantitative determination of morphological changes under silicon-limitation, a hundred randomly selected cells from triplicate cultures were categorized into 4 types (see Results) by their SEM images, and then the mean percentage of each type was calculated. The data was analyzed by t-test.