Af6197

Human fetal eyes were fixed overnight in 4% paraformaldehyde. The samples were then washed in PBS, dehydrated overnight at 4 °C with 20% sucrose solution, embedded in 30% sucrose and OCT (Tissue‐Tek O. C. T. Compound) at a 1:1 ratio, and snap‐frozen in dry ice. Thin (8–10 µm) cryosections were obtained using a cryostat (Leica). For immunohistochemistry, sections were permeabilized for 20 min at 20–28 °C in 0.3% Triton X‐100 (P0096, Beyotime), washed three times in PBS, blocked for 2 h at 37 °C with 10% normal goat serum, and incubated for 16 h at 4 °C with primary antibodies diluted in prefabricated solution. Primary antibodies specific for NRL (1:200; sc‐374277, Santa Cruz), RGR (1:500; ABP56042, Abbkine), CALBINDIN (1:200; 66394‐1‐Ig, Proteintech), PKC (1:200; AF6197, Affinity), CALRETININ (1:200; 92635T, CST), BRN3A (1:200; sc‐8429, Santa Cruz), OPSIN‐Blue (1:300; AB5407, Millipore), OPSIN‐Red/Green (1:300; AB5405, Millipore), and REST (1:200; sc‐374277, Santa Cruz) were used. Subsequently, sections were washed with PBS and incubated for 2 h at room temperature between 20 and 25 °C with secondary antibodies (1:500; A0423/A0521, Beyotime). DAPI (C1006, Beyotime) staining was used to visualize the nuclei. Images were captured using a fluorescence microscope and processed using the ImageJ software.