a–a”, P14 retina section double-immunostained with anti-PlexA4 (a, red) and anti-Sema6A (a’, green) (merged in a”). Strong Sema6A immunoreactivity in the IPL was observed in ~one-half of S3 and throughout S4 and S5, whereas PlexA4 expression is stratified in two distinct layers in S1 and S2.
b, P14 retina section double-immunostained with anti-PlexA4 (green) and anti-calbindin (CB, red) shows PlexA4 protein localization in S1/S2 sublaminae relative to calbindin-positive neuronal processes.
c, P14 retina section double-immunostained with anti-Sema6A (green) and anti-calbindin (CB, red) shows Sema6A protein localization in the S3–S5 relative to calbindin-positive neuronal processes.
d–d”, P14 retina section double-immunostained with anti-PlexA4 (d, green) and anti-TH (d’, red), revealing co-localization of PlexA4 and TH immunoreactivity in S1 of the IPL (merged in d”).
e–e”, P14 retina section hybridized with PlexA4 antisense probe (e, green) followed by anti-TH immunolabeling (e’, red, merged in e”). PlexA4 mRNA is localized to the cell body of dopaminergic amacrine cells (of 26 TH-positive amacrine cells scored, all were positive for PlexA4 mRNA).
f and f’, P14 retina section double-immunostained with anti-PlexA4 (green) and anti-N-terminal melanopsin (red), which labels multiple ipRGC subtypes (f; high magnification of the area in the white square shown in f’). PlexA4 immunoreactivity was not observed in the cell bodies or dendrites of ipRGCs.
g and h, P14 retina sections double-immunostained with anti-Sema6A (green) and anti-TH (g, red) or anti-N-terminal melanopsin (Me) (h, red). Sema6A protein was not observed in cell bodies, or processes, of dopaminergic amacrine cells and ipRGCs (M1-type ipRGC dendritic processes in the S1 indicated by yellow arrow in h).
Scale bars: 50 µm in c for a–a”, b, c, 50 µm in h for d–d”, f, g–h, 20 µm in e” for e–e”, and 20 µm in f’.