Figure 1. Multiple immunofluorescence staining of neuronal markers in adult rat brain sections, as visualized in colour-coded images from confocal laser scanning microscopy. (a) Concomitant visualization of neurons immunoreactive for the GABA_A (-aminobutyric acid type A) receptor ₁ subunit (red) and axons positive for neuropeptide Y (green) in the olfactory bulb. Sections were incubated with a mixture of primary antibodies raised in guinea pig and rabbit, followed by a mixture of secondary antibodies (goat anti-rabbit conjugated to Cy2 and goat anti-guinea pig conjugated to Cy3). Images corresponding to the two fluorochromes were captured simultaneously and are displayed in a pseudo three-dimensional rendering mode to illustrate that the two markers are fully segregated in this section. Bar, 10 m.

(b) Triple immunofluorescence staining for the GABA_A receptor ₁ subunit (green; Cy2), the calcium-binding protein parvalbumin (red; Cy3) and neuropeptide Y (blue; Cy5) in the CA1 region of the hippocampus. Note the pair-wise, partial colocalization of these markers in subsets on neurons (arrowheads). Bar, 20 m.

(c) Triple immunofluorescence staining for the calcium-binding proteins parvalbumin (green; Cy2), calretinin (blue; Cy5) and neuropeptide Y (red; Cy3) in the cerebral cortex. Note that, unlike the hippocampus (b), the three markers are fully segregated in distinct subpopulations of neurons. For panels (b) and (c), signals corresponding to the three fluorochromes were captured simultaneously. The corresponding, colour-coded images were superimposed from a stack of 12 confocal sections. Bar, 20 m.