TEM assays were performed according to the methods described by Leng et al. (2017a (link)). Fresh leaves of wild-type and wls5 plants were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4) for at least 4 h and washed in PBS three times. The samples were then postfixed with 1% (w/v) OsO4 for 2 h after extensive washing in PBS, dehydrated with a graded ethanol series and infiltrated with Spurr Kit (Sigma). The specimens were sectioned (70 nm ultrathin) with a Leica EM UC7 ultramicrotome, and sections were stained with uranyl acetate and alkaline lead citrate for 10 min. TEM was performed using a Hitachi model H-7650.
Spurr kit
Manufactured by Merck Group
The Spurr Kit is a set of laboratory equipment used for the preparation and embedding of samples for electron microscopy. The kit contains the necessary chemicals and supplies to create a resin medium that can be used to support and preserve the structure of biological or inorganic specimens for examination under an electron microscope.
Automatically generated - may contain errors
Lab products found in correlation
Em uc7 ultramicrotome, by Leica (1 mentions)
H 7650, by Hitachi (1 mentions)
Aqueous uranyl acetate, by Merck Group (1 mentions)
Lead citrate, by Serva Electrophoresis (1 mentions)
Sodium cacodylate buffer, by Serva Electrophoresis (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Jem 100cx 2 electron microscope, by JEOL (1 mentions)
Osmium tetroxide, by Serva Electrophoresis (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Ultracut e ultramicrotome, by Leica (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
H 7500, by Hitachi (1 mentions)
4 protocols using spurr kit
1
TEM Analysis of Plant Leaf Ultrastructure
2
Ultrastructural Analysis of Viral Cores
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The GE cells were harvested and fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in PBS (pH 7.4) overnight. Then the samples were post-fixed with 1% osmium tetroxide for 3 hrs, followed by dehydration in an increasing ethanol series of 50%, 75% and 100% (twice). The samples were embedded using the Spurr kit (Sigma), sliced into ultrathin sections (70–90 μm) and stained with 2% uranyl acetate, 1% lead citrate. The ultrathin sections were viewed under the JOEL JEM 2010F electron microscope. For Cryo-EM, Quantifoil grids were freshly glow discharged at 10 mA at 40 s. 4.5 μl of virus was place on a grid. Grids with sample were plunged freeze in liquid ethane37 (link) and viewed under Titan krios 300 KV. The thickness of viral core shell was measured by Image J according to software menu.
3
Ultrastructural Analysis of Embryoid Bodies
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To prepare semi-thin and ultrathin sections, EBs were fixed by 2.5% glutaraldehyde (Sigma-Aldrich) in 100 mM sodium cacodylate buffer (Serva, Germany), pH 7.0 for 24 h at 4°C and then post-fixed with 1% osmium tetroxide (Serva, Germany) in 100 mM cacodylate buffer, pH 7.0 (all from Sigma-Aldrich) within 1 h at 4°C. After dehydration procedures in serial ethanol and acetone solutions and impregnation in resin, the specimens were embedded into the Spurr’s resin (Spurr kit, Sigma) or Araldite (Fluka). The EB sections were prepared using Ultrotome 3 (LKB, Germany). Semi-thin sections were stained with toluidine blue in 1% sodium tetraborate (Sigma-Aldrich). Ultra-thin sections were placed on the grids and stained with aqueous uranyl acetate (Merck, Germany) and lead citrate (Serva, Germany). The sections were examined under a JEM 100CXII electron microscope (Jeol, Japan).
4
Cell Wall Labeling and Ultrastructure Imaging
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For labeling of the cell wall, semi-thin (0.5 mm) sections were directly stained with calcofluor white (Sigma-Aldrich) for 5 min at room temperature, washed three times with phosphate-buffered saline, and subjected to confocal imaging with a Zeiss LSM700 laser scanning microscope. For transmission electron microscopy, intercalary meristem zones of sui1-4 and WT plants were fixed overnight in 2% glutaraldehyde at 4°C, then exposed to 2% w/v OsO4 for 1 h. The samples were dehydrated under 30, 50, 70, 90, and 100% ethanol, infiltrated, and embedded in a low-viscosity medium with the Spurr Kit (Sigma). Samples were cut into ultra-thin sections (90 nm) using an Ultracut E Ultramicrotome (Leica) and mounted on formvar-coated copper grids. After post-staining with uranyl acetate and lead citrate, the sections were observed under a Hitachi H7500 transmission electron microscope.
High-pressure freezing and subsequent immunogold labeling were performed as described previously [66 (link)]. Immunolabeling of ultra-thin serial sections was performed using standard procedures [67 (link)] with JIM7 primary monoclonal antibody (purchased from the University of Georgia, USA) diluted 1:50 and anti-rat IgG-gold antibody (Sigma-Aldrich) diluted 1:20. For negative contrast, anti-PIP1s antibody was purchased from Agrisera.
High-pressure freezing and subsequent immunogold labeling were performed as described previously [66 (link)]. Immunolabeling of ultra-thin serial sections was performed using standard procedures [67 (link)] with JIM7 primary monoclonal antibody (purchased from the University of Georgia, USA) diluted 1:50 and anti-rat IgG-gold antibody (Sigma-Aldrich) diluted 1:20. For negative contrast, anti-PIP1s antibody was purchased from Agrisera.
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