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 bound primary antibodies were detected with alkaline phosphatase-conjugated anti-rabbit or anti-guinea pig IgG secondary antibodies (Tönnes et al., 1999). A series of primary and secondary antibody dilutions and incubation times were used to optimize the experimental conditions for the linear sensitivity range of the alkaline phosphatase reactions. To compare the expression levels of each protein during development, all nitrocellulose membranes were processed in parallel, and the same incubation time for each reagent was used for all antibodies at all ages. Therefore, some regions that showed very low levels of expression may have been considered as negative. For this reason, we only performed quantitative analysis on the expression levels from P5. We only compared labelling intensities obtained with the same antibody.
