To establish the relative abundance of GIRK channel subunits in CA1 pyramidal cells during development, quantification of immunolabelling was carried out in three different ways. (i) To determine the abundance of GIRK1, GIRK2 and GIRK3 immunoreactivity in different compartments of pyramidal cells during development, we used 60 μm coronal slices processed for pre-embedding immunogold immunohistochemistry. The procedure was similar to that used previously (Luján et al., 1996; Luján & Shigemoto, 2006). Briefly, for each of three animals from different postnatal ages and adult, three samples of tissue were obtained for the preparation of embedding blocks (totalling nine blocks for each age). To minimize false negatives, ultrathin sections were cut close to the surface of each block. We estimated the quality of immunolabelling by always selecting areas with optimal gold labelling at approximately the same distance from the cutting surface. Randomly selected areas were then photographed from the selected ultrathin sections at a final magnification of 45 000×. Quantification of immunogold labelling was carried out in reference areas totalling approx. 2000 μm2 for each age. Immunoparticles identified in each reference area
