Figure 5

Immunogold labeling of host proteins in purified infectious bronchitis virus (IBV) particles. Purified IBV particles were either mock treated (A) or subjected to digestion with bromelain (B-F) followed by concentration through a sucrose cushion and then immunogold labeled with antibodies against (A) IBV mock, (B) IBV, (C) normal mouse IgG, (D) Actin, (E) Annexin A2, (F) Hsp90. Labeled virions were fixed in 2% paraformaldehyde for 5 min at RT and treated with Triton X-100 (0.2%) in PBS (pH 7.4) for 5 min and then blocked with 5% BSA in PBS-Tween 20 (pH 7.4) for 30 min at RT. After being incubated with primary and secondary antibody, the virions were negatively stained with 2% sodium phosphotungstate and visualized by electron microscopy (30,000 × magnification). Arabic numbers shown outside the blanket indicate the quantity of gold particles per virion while numbers shown inside the blanket indicate the quantity of virions counted.