Immunocytochemical Detection of β2-AR

After pharmacological treatments, HL-1 cells were washed in PBS and then stained in ice with an anti-β₂-AR antibody (1:100, ab61778, Abcam, Cambridge, UK) for 45 min, following the manufacturer’s instructions. The anti-β₂-AR antibody was chosen based on previously reported data [30 (link),31 (link)]. After washing, cells were incubated with AlexaFluor 488-conjugated anti-rabbit IgG (Invitrogen Corporation, Milan, Italy) as a secondary antibody for an additional 30 min. At the end of staining, cells were fixed in 2% paraformaldehyde for 15 min, counterstained with Hoechst 33,258 (Sigma–Aldrich, St. Louis, MO, USA) at the concentration of 1 mg/mL in PBS, and then mounted in glycerol/PBS (ratio 1:1, pH 7.4). Images were acquired by intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation, Milan, Italy) equipped with a Zeiss charge-coupled device (CCD) camera (Carl Zeiss, Oberkochen, Germany).