Pre Coating Electron Microscope Grids Prior to ISEM 289

1. In a Parafilm-lined Petri dish, place carbon-coated grids, carbon filmside down, onto 15-^L drops of 0.01 mg/mL protein A in distilled water for 5 min at room temperature.

2. Wash the grids with 15 drops of phosphate buffer, pH 6.5, and transfer to 20-^L drops of antiserum diluted 1:50 in phosphate buffer, pH 6.5. Incubate at room temperature for 10 min (see Note 19).

3. Wash grids with 15 drops of phosphate buffer, pH 6.5, drain and transfer to 15-^L drops of sap extract containing virus, diluted in phosphate buffer, pH 6.5, to 1:500. Incubate at room temperature for 30 min.

4. Wash the grids with 20 drops of distilled water and stain with three to five drops of 2% uranyl acetate.

5. To decorate virus particles follow steps 5-7 of Subheading 3.1.2. before staining.

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