HEK 293 cells were grown on poly-L-lysine-coated coverslips overnight. Cells were then transiently transfected with CALCR. After 24 h, cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 30 min at room temperature and washed several times with phosphate buffer. Specimens were permeabilized with ice-cold methanol and then incubated with Anti-Calcitonin receptor /CALCR Rabbit Polyclonal Antibody (Product # 720369 at a concentration of 2 µg/mL. Cells were then incubated with Alexa488-conjugated secondary antibody for 2 h at room temperature, mounted and examined using a laser scanning confocal microscope. Note, strong staining of transfected cells and no staining of adjacent non-transfected cells.