PAG Labeling Without ISEM

1. Grind infected material on a glass slide in phosphate buffer, pH 6.5, to a dilution of 1:10.

2. Place 20-^L drops of sap on Parafilm in a Petri dish.

3. Place carbon-coated grids on the sap, carbon side down, cover with the Petri dish lid, and incubate at room temperature for 15 min.

4. Dilute specific antiserum in buffer to 1:500 and place 20-^L drops on the Parafilm.

5. Take the grids off the sap and place them on the diluted antiserum. Incubate for 15 min as before.

6. Dilute PAG in phosphate buffer to 1:50.

7. Wash grids by holding in the forceps and dropping 30 drops of phosphate buffer in a constant stream using a Pasteur pipet on the treated side. Drain excess liquid with filter paper and place on PAG for 15 min at room temperature.

8. Wash with 20 drops of buffer, five drops of distilled water, and stain with three drops of 2% uranyl acetate, drain excess liquid with filter paper.

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