Briefly, free-floating sections were incubated in 10% (v/v) NGS diluted in TBS. Sections were then incubated in anti-GIRK1, anti-GIRK2 or anti-GIRK3 antibodies [3-5 μg/mL diluted in TBS containing 1% (v/v) NGS], followed by incubation in goat anti-rabbit or anti-guinea pig IgG coupled to 1.4 nm gold (Nanoprobes Inc., Stony Brook, NY, USA). Sections were postfixed in 1% (v/v) glutaraldehyde and washed in double-distilled water, followed by silver enhancement of the gold particles with an HQ Silver kit (Nanoprobes Inc.). Sections were then treated with osmium tetraoxide (1% in 0.1 M phosphate buffer), block-stained with uranyl acetate, dehydrated in graded series of ethanol and flat-embedded on glass slides in Durcupan (Fluka) resin. Regions of interest were cut at 70-90 nm on an ultramicrotome (Reichert Ultracut E, Leica, Austria) and collected on single slot pioloform-coated copper grids. Staining was performed on drops of 1% aqueous uranyl acetate followed by Reynolds’s lead citrate.
