Cell culture, transfection and labeling of HaloTag-Ras

On the day of imaging, coverslips were washed with phosphate buffer saline and cells were labeled with 25pM fluorescent (JF646 or JF549) HaloTag ligand (30 minutes), which covalently binds to the HaloTag-RAS molecules. After incubation with the ligand, cells are washed three times with PBS before replenishing cells with complete medium.  It is a good idea to use complete medium without phenol red, as this can also contribute to background.