H500 transmission electron microscope

For the TEM studies, a morphogenic callus of Brachypodium was fixed in 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (PB; pH 7.4) at 4 °C for 24 h. After washing in phosphate buffered saline (PBS, 5 times, 30 min each), the material was postfixed in 1% osmium tetroxide in a 0.1 M PB (4 °C, 24 h), rinsed with the same buffer and dehydrated in a graded concentration series of ethanol (30%, 50%, 70%, 90%, 96%, and 100%, each for 15 min) and acetone (two times, 15 min each) followed by infiltration in mixtures of acetone and Epon 812 resin (3:1, 1:1, and 1:3) (Polysciences, Eppelheim, Germany). Then, the material was embedded in Epon 812 resin and polymerized into resin blocks at 60 °C for 48 h. Ultra-thin (70 nm) sections were cut with a diamond knife on an Ultracut UCT25 (Leica, Wetzlar, Germany) ultramicrotome. After contrast staining with uranyl acetate and lead citrate, the sections were examined using a H500 transmission electron microscope (Hitachi, Tokyo, Japan).

The fixed material from in vitro culture and fixed in Karnovski's solution fragments of rats’ kidney were rinsed in phosphate buffer to remove the fixative solution and embedded in epoxy resin (Poly/Bed^®812 Embedding Media/DMP-30 Kit, Polyscience, Inc., Warrington, PA, USA) according to the standard methodology used in histological studies^{39 (link)}. The material obtained from in vitro culture was dehydrated in ethanol-acetone series and embedded in epoxy resin directly on the culture plates.
The obtained epone blocks were cut with a Leica Ultracut UCT ultramicrotome. Semi-thin sections were stained with 1% methylene blue solution at high temperature. For TEM analyses, the ultra-thin sections were contrasted with uranyl acetate and lead citrate. Observations were made using a Hitachi H500 Transmission Electron Microscope with an accelerating voltage of 75 kV.
The tissues for histological examination were fixed in Bouin's solution, rinsed in ethanol series and embedded in paraffin blocks. Paraffin sections were stained using AZAN trichrome method (stain used: 0.1 g azocarmine G/100 ml distilled water, 2 g orange G/100 ml distilled water and 0.5 g aniline blue/100 ml distilled water). Staining was performed according to standard histological methodology. Histological preparations were observed using Olympus BX60 microscope.

The ultrastructural changes in fibroblast cells were determined based on TEM analysis. Fibroblasts were treated with NPs at a 50 µg/ml concentration for 24, 48, and 72 h. Then detached by trypsinization, the cell suspension was prepared according to the standard TEM analysis method: washed, dehydrated, and embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma)^{39 (link),40 (link)}. Ultra-thin Sects. (70 nm) were cut on a Leica Ultracut UCT25 ultramicrotome and stained with uranyl acetate and lead citrate. The material was analyzed using a Hitachi H500 transmission electron microscope at 75 kV^{41 (link)}.

Dissected seminal vesicles were fixed in 2.5 % glutaraldehyde in a 0.1 M phosphate buffer (pH = 7.4) for 24 h at room temperature. After fixation, the material was rinsed several times with a mixture of 50 ml 0.1 M phosphate buffer and 50 ml of ddH₂O to which 4.6 g saccharose was added. Afterwards, the material was postfixed for 2 h in a mixture of 1 % OsO₄ in a 0.1 M phosphate buffer, dehydrated in a graded series of ethanol replaced by acetone and then embedded in an Epoxy Embedding Medium Kit (Sigma, St. Louis, MO, USA). Semi-thin sections (1 μm thick) were stained with methylene blue and then examined under an Olympus BX60 microscope equipped with an XC50 digital camera and CellSense Standard software. After contrasting with uranyl acetate (15 min) and lead citrate (20 min), ultra-thin sections (70 nm thick) were examined using a Hitachi H500 transmission electron microscope.

Samples of the root tip (4 mm) were fixed for 4 h at room temperature in a 2.5% solution of glutaraldehyde in 0.1 M Na-phosphate buffer, pH 7.2, containing sucrose (15 mg/mL). After washing in the same buffer, probes were post-fixed using 1% OsO₄ (Sigma, St. Louis, MO, USA) on ice for 2 h. Later, the material was dehydrated and embedded into Epoxide resin (Epon-812 and Araldite mixture, Merck, Darmstadt, Germany) using a standard technique [73 (link)]. Ultrathin sections were prepared with an LKB-III microtome (LKB, Stockholm, Sweden). The ultra-thin (1–2 μ) sections were contrasted with lead citrate and viewed using an H-500 transmission electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 10 kV and operating magnification of 10,000×. For chromatin morphology statistical analyses, we measured the area of the nucleus using the Cell program. Its darkly contrasted and electron-transparent chromatin areas correspond to the dense and loose chromatin, respectively. We used more than 30 photos with cell 60 nuclei of the interphase nucleus of meristem of progeny stem cells. The parameters were measured using a BX51 microscope (Olympus, Tokyo, Japan) furnished with the Cell program.