Immunofluorescence

Transiently transfected COS7 cells were seeded onto glass coverslips and cultured in incubator overnight. Cells were washed 5 min for three times with PBS and fixed with 4% paraformaldehyde for 20 min. Then fixed cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min and blocked with 5% BSA at room temperature for 1 h. After blocking, the samples were incubating with anti-Myc rabbit polyclonal antibody (1:2000), anti–protein disulfide isomerase (PDI) mouse monoclonal antibody (1:200) or anti-GM130 mouse monoclonal antibody (1:200) respectively at 4℃ overnight. Next day, cells were washed with PBS three times and then incubated with secondary antibody conjugated with different Alexafluor dyes at room temperature for 1 h (away from light). Finally, fluorescent images with a × 40 objective were collected under fluorescent microscope using Axio Imager M2 (Carl Zeiss, Oberkochen, Germany).