solution for the second antigen and the procedure for conjugation of the fluorophore-conjugated secondary antibody was repeated as above. Finally, cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and washed twice before imaging using a fully automated epifluorescence microscope equipped with a 20× Fluor objective and a Photometrics CoolSNAP HQ 12-bits digital interline CCD camera (ImageXpress Micro, Molecular Devices, Sunnyvale, CA). For each condition, 4 wells were imaged with 6 different sites per well. Cells cultured on glass coverslips were mounted on glass slide with Prolong antifade reagent (Invitrogen) and imaged using a Zeiss Axio Observer Z1 inverted microscope.
