EdU detection was performed using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen). Sections were permeabilized with 0.2% Triton X-100 (Progen Industries) in dPBS for 30 min, washed with 3% protease-free BSA (Sigma-Aldrich) in dPBS (2 × 5 min), then incubated with 300 μl reaction mixture prepared according to kit directions for 30 min. In the first pilot run, cells were not double labeled with Brn3a to distinguish RGCs, because at P0 there are as yet no displaced amacrine cells in the GCL (Perry, 1981 (link)). In the more extensive follow-up experiment we also wished to obtain laser-captured E18 RGCs from P5 retinas at a time when their axons are in the process of innervating the SC; however, at this age potentially EdU+ amacrine cells are now present in the GCL. Thus for all groups in this second series we additionally immunostained retinal sections for Brn3a protein, an established marker for RGCs projecting to the SC (Nadal-Nicolás et al., 2009, 2012 (link)). Retinal sections were washed with dPBS (2 × 10 min) and incubated overnight at 4°C with anti-Brn3a goat primary antibody (AB; Santa Cruz Biotechnology, SC-31984) 1:100 in antibody diluent (10% normal horse serum and 0.2% Triton X-100 in dPBS). After washes, sections were incubated in donkey anti-goat Cy3 (Jackson ImmunoResearch, 705-166-147; 1:200 in antibody diluent) for 2 h.
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