Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus

Wang, Yuan; Li, Yan; Ding, Tianbing

Confocal laser scanning microscopy (CLSM). Routine CLSM was performed (29) to detect the location of HSP90β-JEV binding. Briefly, Vero cells were prepared on glass coverslips, fixed by paraformaldehyde, and incubated with JEV (1.2x10 5 PFU/ml) at 37˚C for 1 h. After washing 3 times with PBS, a primary antibody (1:10 anti-JEV mAb 2H4, and 1:50 rabbit anti-human HSP90β mAb, respectively) was added followed by incubation overnight at 4˚C, and the secondary antibody [1:100 FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich™ Cat. no. F9006), and 1:100 Cy3-conjugated goat anti-rabbit IgG (Sigma-Aldrich™ Cat. no. C2306), respectively (Sigma-Aldrich)] was added followed by incubation in the dark at room temperature for 1 h followed by a PBS wash 3 times. The coverslips were then thoroughly rinsed in deionised water, air dried, placed reversely on a glass slide mounted with mixture of 50% glycerol and an equal volume Hoechst (1:1,000; Beyotime Institute of Biotechnology, Shanghai, China), and the samples were finally observed under an Olympus FV-1000 microscope with the image analysis software package FV1000 Viewer v1.4a (Olympus Corp; Tokyo, Japan).