PAG Labeling Using Mixed Virus Antisera

1. Using a variable volume pipet, dilute two (or more) specific antisera to 1:500 in phosphate buffer, pH 6.5, in 0.5-mL microfuge tubes.

2. In a Parafilm-lined Petri dish, place 20-pL drops of the diluted antiserum and, using the watchmaker's forceps, float carbon coated grids, carbon side down, on them. Incubate at room temperature for 15 min.

3. Grind infected material of two (or more) antigens on a glass slide in phosphate buffer, pH 6.5, to a dilution of 1:10.

4. Place 20-pL drops of sap on Parafilm. Wash grids as before and place carbon-coated grids on the sap, carbon side down, replace the Petri dish lid, and incubate at room temperature for 15 min.

5. Select the first specific antiserum and dilute in phosphate buffer pH 6.5 to 1:500 in a centrifuge tube. Place 20-pL drops on the Parafilm.

6. Wash grids as before and place them on the antiserum for 15 min.

7. Dilute a small size of PAG particle, for example, 5 nm, in phosphate buffer, pH 6.5, to 1:50.

8. Wash grids as before. Drain excess liquid with filter paper and place on the PAG for 15 min.

9. Select a second specific antiserum and dilute as before.

10. Wash grids as before and place them on the second antiserum for 15 min.

11. Dilute a larger size of PAG particle, for example, 20 nm, wash grids as before, and place them on the PAG and incubate for 15 min as before.

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

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