Sections were incubated in 10% (v/v) normal goat serum (NGS) diluted in 50 mm Tris buffer (pH 7.4) containing 0.9% (v/v) NaCl [Tris-buffered saline (TBS)], with 0.2% (v/v) Triton X-100 for 1 h. Sections were then incubated for 48 h in either anti-GIRK1, anti-GIRK2 or anti-GIRK3 at a final protein concentration of 1–2 μg/mL diluted in TBS containing 1% (v/v) NGS. After several washes in TBS, the sections were further incubated for 2 h in biotinylated goat anti-rabbit IgG or anti-guinea pig IgG (Vector Laboratories, Burlingame, CA, USA) diluted 1 : 100 in TBS containing 1% (v/v) NGS. They were then transferred into avidin–biotin–peroxidase complex (ABC kit; Vector Laboratories), diluted 1 : 100, for 2 h at room temperature. Bound peroxidase enzyme activity was revealed using 3,3′-diaminobenzidine tetrahydrochloride (0.05% in Tris buffer, pH 7.4) as the chromogen and 0.01% (v/v) H2O2 as the substrate. Finally, sections were air-dried and coverslipped prior to observation in a Nikkon photomicroscope (Nikkon, Eclipse 80i) equipped with differential interference contrast optics and a digital imaging camera.
