4 μl of purified CCV preparation (0.33 mg/μl) was applied to carbon-coated and glow discharged (2 min at 7×10-1 mbar) 300-mesh copper EM grids (Electron Microscopy Sciences; CF300-CU) and incubated for 4 min at room temperature. Excess solution was removed with blotting paper and samples immediately fixed by a 20 min incubation at room temperature with 2% glutaraldehyde (v/v) in PEM buffer (100 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.9). Samples were then washed with phosphate buffer (0.1 M, pH 7.4) and distilled water, followed by a 20 min incubation with 0.1% tannic acid (w/v) in MilliQ water. After three washes in water, the samples were incubated with 0.2% aqueous uranyl acetate for 30 min at room temperature and then washed three times with water before being dehydrated in a series of graded ethanol (10%, 20%, 40%, 60%, 80%, 96% and 100% for 2 min each) and two washes with hexamethyldisilane (99%). Dried samples were then coated with 3 nm platinum and 4 nm carbon using an ACE600 coating device (Leica Microsystems). The sample grids were imaged using a JEOL JEM2800 scanning/transmission electron microscope at 200 kV.
Ace600 coating device
Manufactured by Leica
The ACE600 is a coating device manufactured by Leica. It is designed to apply thin films or coatings to various substrates. The device utilizes a vacuum deposition process to deposit materials onto the target surface. The ACE600 is a versatile tool suitable for a range of applications that require precise and controlled film deposition.
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Lab products found in correlation
2 protocols using ace600 coating device
1
Cryo-Electron Microscopy Specimen Preparation
2
Protoplast Extraction and SEM Preparation
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The plated protoplasts were washed in PBS equilibrated to room temperature and the extraction solution (1% Triton X-100 in PIPES-EGTA-magnesium buffer (PEM; 100 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9) plus 1% high-MW polyethylene glycol (PEG; 20 kDa) and 2 µM phalloidin ±2 µM taxol) was applied for 4 mins. Samples were then washed three times in PEM plus 1% PEG for 1 min and then fixed in 2% Glutaraldehyde in phosphate buffer (PB) for 20 mins. After washing in distilled water, samples were treated with 0.1% tannic acid in water (w/v) for 20 mins at room temperature and 0.2% uranyl acetate in water (w/v) for 20 mins at room temperature. Samples were then ‘critical point dried’ as described by Svitkina (2007) (link) and colleagues. Samples were then fixed on SEM specimen mounts by means of carbon conductive adhesive tabs (Ø 12 mm), and gold-coated to a thickness of 5 nm by rotary shadowing at a 45° angle using a ACE600 coating device (Leica Microsystems).
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