Sc 271322

Three coronal slices of 250-μm thickness of the aIC (AP, +2.5 to +1.7 mm) and pIC (AP, −1.7 to −2.5 mm) of HC control rats were cut on a cryostat. Bilateral punches of the agranular aIC and pIC (thus six punches per region of interest) were acquired with a 1.0-mm brain puncher and snap frozen in isopentane on dry ice. Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred on polyvinylidene difluoride membranes (Bio-Rad, 170–4156), and probed with antibodies against the β₁-adrenoceptor (Rabbit, 1:1,000, Invitrogen PA1-049) and β₂-adrenoceptor (Mouse, 1:100, Santa Cruz, sc-271322) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000, Cell Signaling #2118) for normalization. Proteins were then detected with horseradish peroxidase–conjugated Goat anti-Mouse (1:50,000, Jackson ImmunoResearch Laboratories, 115–035-062) and Goat anti-Rabbit (1:50,000, Invitrogen, G21234). Proteins were revealed with Super Signal West Femto ECL (Thermo Fisher Scientific, 34095) and visualized with ChemiDoc Touch Imaging system (Bio-Rad).

Measurements of immunofluorescence were performed as previously described [25 (link)]. The cells were incubated with the indicated primary antibodies against β2AR (1:100 dilution, sc-271,322, Santa Cruz) and Rab11 (1:100 dilution, #5589, Cell Signaling Technology) overnight at 4 °C and then with Alexa Fluor 488-conjugated anti-mouse IgG (sc-516,140, Santa Cruz) or Alexa Fluor 647-conjugated anti-rabbit IgG (ab150075, Abcam, Cambridge, MA, USA) in the dark at room temperature for 1 h. The stained cells were observed and captured using a laser-scanning confocal microscope (LSM510, Carl Zeiss, Inc.).