ER-Tracker and Immunofluorescence Staining

ER-Tracker Red kit (C1042S) was purchased from Beyotime. Cells were cultured in 24-well plate containing glass coverslips. After 48 h of transfection of SCARA5 plasmid, diluted ER tracker was added and incubated for half an hour in 37°C. Washed cells for 5 min, and fixed with 4% paraformaldehyde for 30 min, permeabilized for 5 min in 0.5% Triton X-100,and blocked with 5% BSA for 1 h at 25°C. Samples were incubated overnight with primary antibody of SCARA5 (#ab118894, abcam) at 4°C. The next day, washed the cells with PBS for three times (5 min once) and incubated with Alexa 488-conjugated goat anti-rabbit secondary antibody for 1 h (Avoid light). Nuclei were stained with DAPI (Roche, Palo Alto, CA, USA). Picked out the coverslip, added 4 μl anti-quenching agent, and covered the slide onto a glass observed with Laser scanning confocal microscope.