The regional distribution of GIRK channel subunits was analysed in rodent brains, using an in-situ blotting technique (histoblot) (Tönnes et al., 1999). For this technique, the expression patterns for GIRK1 and GIRK2 were determined in mouse brains, whereas that for GIRK3 was determined in rat brains; a number of attempts to detect GIRK3 in mouse brains did not yield reliable labelling (Supporting Information Fig. S1). Briefly, horizontal cryostat sections (10 μm) from mouse or rat brain were apposed to nitrocellulose membranes moistened with 48 mM Tris-base, 39 mM glycine, 2% (w/v) sodium dodecyl sulphate and 20% (v/v) methanol for 15 min at room temperature (~20 °C). After blocking in 5% (w/v) non-fat dry milk in phosphate-buffered saline, nitrocellulose membranes were treated with DNase I (5 U/mL), washed and incubated in 2% (w/v) sodium dodecyl sulphate and 100 mM β-mercaptoethanol in 100 mM Tris–HCl (pH 7.0) for 60 min at 45 °C to remove adhering tissue residues. After extensive washing, the blots were reacted with affinity-purified anti-GIRK1, anti-GIRK2 and anti-GIRK3 antibodies (0.5 mg/mL) in blocking solution overnight at 4 °C. The
