click-imaging

Following DPBS wishing, the cells were subjected with reaction buffer for click coupling (1.1 mM CuSO4, 10 mM sodium ascorbate, 15 µM Cy5-azide in 50 mM HEPES buffer with pH 7.6) for 30 min, followed by three times washing with DPBS. Then primary antibody ( Anti-Halo rabbit polyclonal antibody for Halo detection with 1:1000 dilution in DPBS, anti-Calnexin mouse monoclonal antibody for ER staining with 1:200 dilution in DPBS for 2 h at room temperature. Cells were rinsed three times with DBPS with 5 min incubation for each time, and subjected to second antibody ( anti-rabbat IgG Alex Fluor 488 and anti-ms IgG Alex Fluor 568, with 1:2000 dilution in DPBS) for 1 h at room temperature. Cells were again rinsed three times with DBPS and DPBI (2 µg/mL in DPBS) was treated for 5 min, followed by final rinsing with DPBS. Images were taken using a Nikon spining disk confocal microscope with 100X objective.