Vero E6 cells were fixed for at least 1 h with glutaraldehyde 2.5% in 0.1M sodium cacodylate buffer. Cells were then incubated with anti-vimentin (V9) antibody (Santa Cruz Biotechnology) followed by a secondary goat anti-mouse IgG (H + L) antibody coupled to 10 nm-gold particles (Aurion). For resin embedding, cells were washed three times with a mixture of 0.2M saccharose/0.1M sodium cacodylate. Cells were post-fixed for 1 h with 1% OsO4 diluted in 0.2M Potassium hexa-cyanoferrate (III)/0.1M sodium cacodylate solution. After three 10 min washes with distilled water, the cells were gradually dehydrated with ethanol by successive 10 min baths in 30, 50, 70, 96, and 100% ethanol. Substitution was achieved by successively placing the cells in 25, 50, and 75% Epon solutions for 15 min. Cells were placed for 1 h in 100% Epon solution and in fresh Epon 100% over-night under vacuum at room temperature. Polymerization occurred with cells in 100% fresh Epon for 72 h at 60°C. Ultrathin 70 nm sections were cut using a UC7 ultramicrotome (Leica) and placed on HR25 300 Mesh Copper/Rhodium grids (TAAB, United Kingdom). Sections were contrasted according to Reynolds.60 (link) Images were obtained by Tecnai G2 TEM (Thermo-Fischer/FEI) operated at 200 keV equipped with a 4096 × 4096 pixels’ resolution Eagle camera (FEI).
Hr25 300 mesh copper rhodium grids
Manufactured by TAAB
Sourced in United Kingdom, Germany
The HR25 300 Mesh Copper/Rhodium grids are a type of laboratory equipment used for sample preparation in electron microscopy. These grids provide a support structure for thin samples, allowing for their effective imaging and analysis under an electron microscope.
Automatically generated - may contain errors
3 protocols using hr25 300 mesh copper rhodium grids
1
Immunogold Labeling of Vimentin in Vero E6 Cells
2
Dental Plaque and GCF Sample Embedding
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The dental plaque and GCF paper point samples were fixed with 4% paraformaldehyde in 0.1‐M sodium cacodylate buffer for 5 h at 4°C. Resin embedding was microwave‐assisted with a PELCO BiowavePro+ (Ted Pella). After rinsing twice with a mixture of 0.2‐M saccharose/0.1‐M sodium cacodylate and once with distilled water, samples were gradually dehydrated by successive baths in 50%, 70% and 96% ethanol. Substitution with medium grade LR–White resin (Polysciences) was achieved by two incubations with a mixture of 100% LR–White resin and 96% ethanol in a 2:1 ratio, two incubations with 100% LR–White resin, and completed with samples in 100% LR–White resin. Resin heat‐curing was achieved by polymerization for 72 h at 60°C. All solutions used above were 0.2‐μm filtered. Ultrathin 100 nm sections were cut using a UC7 ultramicrotome (Leica Microsystems) and placed on HR25 300 Mesh Copper/Rhodium grids (TAAB).
3
Wastewater Sample Preparation for TEM Imaging
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The suspension from the concentrated wastewater (200 µL) was fixed with 4% paraformaldehyde in 0.1-M sodium cacodylate buffer for five hours at 4 °C. Resin embedding was microwave-assisted with a BiowavePro+ (Pelco, Fresno, CA, USA) (Supplemental Table S1 ). After rinsing tow times with a mixture of 0.2-M saccharose/0.1-M sodium cacodylate and once with distilled water, samples were gradually dehydrated by successive baths in 50%, 70%, and 96% ethanol. Substitution with LR–White resin (medium grade; Polysciences, Warrington, PA, USA) was achieved by two incubations with a mixture of 100% LR–White resin and 96% ethanol in a 2:1 ratio, two incubations with 100% LR–White resin, and completed with samples in 100% LR–White resin. Resin heat-curing was achieved by polymerization for 72 hours at 60 °C. All solutions used above were 0.2-µm filtered. Ultrathin 100-nm sections were cut using a UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and placed on HR25 300 Mesh Copper/Rhodium grids (TAAB, Aldermaston, England).
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