ISEM see Note 1

This involves coating or "activating" a filmed grid with a specific antiserum, loading with an extract of virus-infected material and, finally, staining. The antiserum concentrates the virus particles and markedly increases the amount visible on the treated grid (10). This technique is especially useful for the detection of viruses that may be low in concentration, or restricted to limited areas, within the plant.

1. Using a variable volume pipet dilute required specific antiserum (see Note 2) to 1:500 (see Note 3) in 0.06 M phosphate buffer pH 6.5 (see Note 4) in a 0.5-mL microfuge tube.

2. In a Parafilm lined Petri-dish place 20-^L drops of the diluted antiserum (see Note 5) and, using the watchmaker's forceps, float carbon-coated grids (see Note 6), carbon-side down, on them. Incubate at room temperature for 15 min (see Notes 7 and 8).

3. Grind infected material (see Note 9) in a muslin-lined bag in phosphate buffer, pH 6.5, with a hand grinder (see Note 10) to a dilution of 1:10.

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

5. Hold the antiserum coated grids in the forceps and wash with 20 drops of phosphate buffer, in a constant stream using a Pasteur pipet, on the treated side (see Note 11). Drain excess liquid with filter paper and place on sap for 15 min at room temperature (see Note 7).

Leukocyte Adhesion Deficiency
Fig. 1. Techniques involved in IEM.

6. Wash the grids with 20 drops of distilled water and stain with three to five drops of 2% uranyl acetate (see Note 12), draining excess liquid as before (see Notes 13 and 14).

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