We used the histoblot technique to determine the regional distribution and expression levels of GIRK channel subunits in the brain during postnatal development and in adults. This method is a reliable and convenient way to compare the regional distribution of different proteins in brain samples without compromising the integrity of antibody-binding sites by tissue fixation, which is required for conventional immunocytochemistry (Jo et al., 2006; Kopniczky et al., 2005; Tönnes et al., 1999). Proteins transferred to nitrocellulose membranes were immunostained with the purified GIRK channel subunit-specific antibodies using conventional immunoblotting. In adult brain (P60), the overall expression patterns of GIRK1, GIRK2 and GIRK3 subunits were rather similar (Figs 1A and 2A), with strong immunoreactivities in the neocortex, cerebellum, hippocampus and thalamus (Figs 1B and C, and 2B). Faint staining was observed in midbrain nuclei, including the inferior and superior colliculus, and brainstem nuclei (Figs 1A and 2A). Very weak staining was observed in basal ganglia nuclei such as the caudate putamen and globus pallidus (Figs 1A and 2A).
