Fully expanded leaf blades of 4-week-old seedlings were collected in the morning. For each line, three seedlings were selected and the middle parts of the leaf blades were cut into small segments and used immediately for microscopic observation. The extent of starch accumulation was determined by iodine staining of the rest (tips and bottom parts) of the sampled leaves. The small segments of leaf blades were fixed in 4% (w/v) paraformaldehyde and 2% (w/v) glutaraldehyde in 50 mM phosphate buffer (pH 7.2) for 24 h and post-fixed with 2% (w/v) osmium tetroxide aqueous solution in the same buffer for 2 h at 4°C. Dehydration and resin infiltration were performed in a graded series of acetone solution and propylene oxide. The samples were embedded in Spurr’s epoxy resin (Sigma-Aldrich, St. Louis, MO, USA). Ultrathin transverse sections were prepared with an ultramicrotome (Ultracut UTC, Leica, Germany) and stained with uranyl acetate and lead citrate, and observed using a transmission electron microscope (JEM-1010, JEOL Ltd., Tokyo, Japan) operated at 100 kV. Semithin transverse sections (1 μm thickness) were subjected to periodic acid–Schiff (PAS) staining and observed using a light microscope (Eclipse Ti-S, Nikon, Tokyo, Japan).
Ultracut utc
Manufactured by Leica
Sourced in Germany
The Ultracut UTC is a high-performance ultramicrotome designed for the preparation of ultra-thin sections for electron microscopy. It features precision cutting and advanced controls to ensure consistent, high-quality sample preparation.
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Lab products found in correlation
3 protocols using ultracut utc
1
Ultrastructural Analysis of Leaf Starch Accumulation
2
Ultrastructural Analysis of Macrophages
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The macrophages were fixed in Karnovsky Fixative for 1 h, washed in 0.1M Na cacodylate buffer pH 7.4 and post-fixed in 1% osmium tetroxide. They were in-block stained with uranyl acetate, then dehydrated and embedded in Embed 812 (Electron Microscopy Sciences). Ultrathin sections were cut on a Leica Ultracut UTC, then stained with uranyl acetate and Reynold’s lead. Images were taken on a Tecnai Biotwin Electron Microscope. For detailed information on the immunogold labeling, please refer to the online supplemental experimental procedures.
3
Transmission Electron Microscopy Sample Preparation
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Samples were prepared as described previously. 8, 10 Briefly, samples were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) at 4°C overnight, washed 3 times with 0.1 M PB for 30 minutes each wash, and postfixed with 2% osmium tetroxide in 0.1 M PB at 4°C for 2 hours. The sections were then dehydrated in 50% and 70% ethanol solutions for 10 minutes each at 4°C, 90% ethanol for 10 minutes at RT, and then 3 times in 100% ethanol for 10 minutes each at RT. The samples were treated with propylene oxide twice for 30 minutes each, placed in a 70:30 mixture of propylene oxide and resin (Quetol-812; Nisshin EM Co., Tokyo, Japan) for 1 hour, and then transferred to fresh 100% resin, followed by polymerization at 60°C for 48 hours. The polymerized resin was sectioned at 70 nm with a diamond knife using an ultramicrotome (Ultracut UTC; Leica, Vienna, Austria). The sections were mounted on copper grids and stained with 2% uranyl acetate at RT for 15 minutes, followed by secondary staining with a lead stain solution (Sigma-Aldrich Co., Tokyo, Japan) at RT for 3 minutes. The grids were observed under a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV.
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