Alexa fluor 647 red conjugated secondary antibodies

HEK293 cells grown on coverslips were transiently transfected with pEGFP-APJ and pcDNA3.1-B2R and fixed after 24 h as previously described [21, 27] . Nonspecific binding was blocked by incubation with 3% bovine serum albumin prepared in PBS pH7.4 for 1 h at room temperature, and then permeabilized for 30 min with 0.01% Triton-X-100 in PBS. Cells were washed three times with PBS and incubated with an anti-B2R monoclonal antibody (1:200) in PBS at 4°C overnight. Cells were washed three times with PBS, followed by incubation with Alexa-Fluor®647 (red)-conjugated secondary antibodies (1:400) (Molecular Probes,

HEK293 cells grown on coverslips were transiently transfected with pEGFP-B2R and pcDNA3.1-KOR and fixed after 24 h as previously described [33] . Non-specific binding was blocked by incubation with 3% bovine serum albumin prepared in phosphate-buffered saline (PBS) containing 0.01% Triton X-100 for 1 h at room temperature. Cells were washed three times with PBS and then incubated overnight with an anti-KOR monoclonal antibody (1:200) in PBS at 4°C. Cells were washed three times with PBS, followed by incubation with Alexa-Fluor ® 647 (red)-conjugated secondary antibodies (1:400) (Molecular Probes, Invitrogen) for 1 h at room temperature. After a final wash with PBS, cells were examined using a Leica DMRE laser scanning confocal microscope (Leica, Milton Keynes, UK).