715 166 150 red

HEK-293T cells were seeded on glass coverslips in 12-well plates. Twenty-four hours later, cells were transfected with CB₂R-YFP cDNA (1 μg) and/or OX₁R-Rluc cDNA (1 μg). Forty-eight hours after, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine before permeabilization with PBS-glycine containing 0.2% Triton X-100 (5 min incubation). Cells were blocked during 1 h with PBS containing 1% bovine serum albumin. HEK-293T cells were labeled with a mouse anti-Rluc antibody (1/100, MAB4400, Millipore, Merck, Darmstadt, Germany) and subsequently treated with a Cy3-conjugated anti-mouse (1/200, 715-166-150 (red), Jackson ImmunoResearch, St. Thomas Place, UK) IgG secondary antibody (1 h each). The CB₂R-YFP expression was detected by the YFP’s own fluorescence. Nuclei were stained with Hoechst (1/100 from stock 1 mg/mL; Sigma-Aldrich). Samples were washed several times and mounted with 30% Mowiol (Calbiochem). Images were obtained in a Zeiss LSM 880 confocal microscope (ZEISS, Germany) with the 63X oil objective.

Transfected HEK-293T cells expressing A₃R–YFP and/or A_2AR–Rluc were treated with vehicle, with 100 nM CGS 21680, or with 100 nM IB-MECA for 30 min. Then, cells were fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 20 mM glycine before permeabilization with the same buffer containing 0.2% Triton X-100 (5 min incubation). Cells were treated for 1 h with PBS containing 1% bovine serum albumin, labeled with a mouse anti-Rluc (1/100, MAB4400, Millipore, Merck, Darmstadt, Germany) antibody, and subsequently treated with a Cy3-conjugated anti-mouse (1/200, 715-166-150 (red), Jackson ImmunoResearch, St. Thomas Place, UK) IgG secondary antibody (1 h each). Samples were washed several times and mounted with 30% Mowiol (Calbiochem, Merck, Darmstadt, Germany). Samples were observed under a Leica SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany).