C2 Immunostaining of Virally Infected CEF or QT6 Cells

The monoclonal antibody (MAb) 3C2 (18) recognizes the viral GAG protein and can be used to assess the extent of infection following transfection of line 0 CEF cells (Subheading 3.3.) and/or to determine the viral titer of concentrated supernatant (Subheading 3.4.).

1. Rinse cells twice with 1X PBS.

2. Fix for 15-30 min at room temperature with 3.5% (v/v) paraformaldehyde in PBS.

3. Rinse cells twice with 1X PBS.

4. Block 3X 30' at room temperature in CEF media containing 0.05% Triton X-100.

5. Incubate in 3C2 1° antibody (diluted 1:4 in CEF plus 0.05% Triton X-100) overnight at 4°C. The 1° antibody can be reused several times, and between uses should be kept at 4°C in the presence of 0.02% (w/v) sodium azide.

6. Rinse 3X 30' with 1X PBS containing 10% serum (e.g., goat serum).

7. Incubate in 2° antibody (peroxidase-conjugated antimouse IgG, IgM diluted 1:200 in 1X PBS plus 10% serum) for at least 1 h at room temperature.

9. Develop with activated DAB. To enhance the signal, 0.5 mg/mL nickel chloride can be added to the DAB solution giving a black precipitate, which is easier to visualize (see Fig. 1).

10. The reaction is stopped by rinsing several times with 1X PBS.

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