Figure 2 shows a single optical section through the ganglion cell layer of a retina incubated in monoclonal anti-HCN4 primary and DyLight 488-conjugated secondary (
Figs. 2A,
2C), anti-Thy1 primary and DyLight 549-conjugated secondary (
Figs. 2B,
2C), and anti-Brn3a primary and DyLight 649-conjugated secondary (
Fig. 2C). The fluorescence emissions from DyLight 488, 549, and 649 were assigned to the green, red, and blue color channels, and are merged in
Figure 2C. Yellow in
Figure 2C shows colocalization of HCN4- and Thy1-like immunoreactivities along the membranes of several somatic profiles. The condensed intracellular blue signal identifies two of these cells (one each at the upper left and far right of the field) as retinal ganglion cells. Colocalization of Thy1 and HCN4 in single retinal ganglion cells is thus shown by the green and red pixels circumscribing the optically sectioned profile of cells with blue pixels in their nuclei.
Figures 2A–C also show HCN4- and Thy1-like immunoreactivities in a nerve fiber layer fascicle (upper right), and one large soma (along the lower edge of the field) without detectable Brn3a-like immunoreactivity. These stainings are compatible with previous descriptions of HCN4-immunopositive, dextran-backfilled somata, and intraretinal nerve fiber bundles.
35 Figures 2D–F show a single optical section through the ganglion cell layer of a different portion of the same retina, processed as in
Figures 2A–C except that the anti-Thy1 primary was not applied. The lack of DyLight 549-fluorescence in
Figures 2E and
2F show that the colocalization in
Figure 2C is not due to binding of both secondary antibodies to the anti-HCN4 primary. Some of the somatic profiles in
Figures 2C and
2F bind both anti-Brn3a and anti-HCN4 antibodies, while a few present HCN4-like immunoreactivity without Brn3a-like immunoreactivity (consistent with the results of Oi et al.
35 ). Results similar to those illustrated in
Figure 2 were obtained in a total of four separately processed retinas.