Indirect Immunocytochemistry for PaBV-4 N Protein

Indirect immunocytochemistry assay, to detect PaBV-4 nucleoprotein (N protein), was conducted similar to a previously described method [4 (link)]. Briefly, the DEF cells were washed twice for 5 min each time in 0.02 M PBS, fixed for 10 min in 2% paraformaldehyde in 0.02 M PBS, and then washed twice for 5 min each time in 0.02 M PBS. Cells were permeabilized using 1% Triton X-100/0.02 M PBS for 10 min and then washed 3 times for 5 min each time in 0.03% Tween/0.02 M PBS. Blocking was performed for 2 hours in 5% dried milk/0.03% Tween/0.02 M PBS. The primary antibody, chicken IgG anti-PaBV-4 N protein, at a 1:500 dilution in 1% dried milk/0.03% Tween/0.02 M PBS, was added to the cells and then incubated in a humidified chamber for 30 min at 37°C.
Cells were washed 3 times for 5 min each time in 0.03% Tween/0.02 M PBS. Secondary antibody, horseradish peroxidase conjugated rabbit anti-chicken IgG (Sigma-Aldrich) at a 1:500 dilution in 1% dried milk/0.03% Tween/0.02M PBS, was added to the cells and incubated in a humidified chamber for 30 min at 37°C. Cells were washed 3 times for 5 min each time in 0.03% Tween/0.02M PBS and then rinsed in distilled water.