The infection of primary avian tracheal epithelial cells with infectious bronchitis virus

Shen, Ching-I; Wang, Ching-Ho; Liao, Jiunn-Wang; Hsu, Tien-Wang; Kuo, Shu-Ming; Su, Hong-Lin

Cells or cryosections of infected trachea were fixed in 4% cold paraformaldehyde and permeabilized with 0.3% Triton-X 100. Immunocytochemistry was performed with the following primary antibodies: E-cadherin (1:500, BD Biosciences, Franklin Lakes, NJ, USA), b-tubulin IV and pan b-tubulin (1:100, Sigma), ZO-1 (1:100, Zymed-Invitrogen), mucin 5AC (1:50, Abcam, Cambridge, MA, USA), cytokeratin 14 (1:100, Convance, Princeton, NJ, USA), smooth muscle actin (SMA, 1:50, DakoCytomation, Glostrup, Denmark) and vimentin (1:500, Convance). Immune-serum of IBV-infected chicken, screened by ELISA, was used for the detection of IBV antigens in infected cells (1:200) [30] . Fixed cells were washed twice with 0.1% Tween-20 in phosphate buffer saline (PBS). Appropriate fluorescence-tagged secondary antibodies (all from Jackson ImmunoResearch, West Grove, PA, USA) were used for visualization. Blue 4 0 ,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counter-staining. Images of immunostaining were captured using a fluorescent microscope (Nikon ECLIPSE 80I) or confocal microscope (LSM510 Meta, Zeiss).