Goat anti s opsin

Criteria for dissection quality were applied before proceeding. Any retina that was appreciably torn in such a way as to mimic the appearance of a reliving cut, or any retina that exhibited significant tissue loss or damage of the central or mid-central retina was discarded. To observe the s-opsin gradient, dissected retinas (Figure 1d – left and middle panel) were mounted on nitrocellulose membrane paper and immunohistochemistry was conducted similarly to Sondereker et al. (2017) (link). Retinas were fixed in 4% paraformaldehyde for 40 minutes and then rinsed (3 × 15 minutes) in 0.1M phosphate-buffered saline (PBS). Retinas were placed in blocking solution and incubated at 4° C overnight. Retinas were then incubated at 4° C in blocking solution for six days with primary antibody goat anti-s-opsin (Santa Cruz Biotechnologies, Cat# sc14363, RRID: AB_2158332). The retinas were washed (6 × 10 minutes) in 0.1M PBS and placed in blocking solution overnight at 4° C with secondary antibody donkey anti-goat Alexa Fluor 594 (Life Technologies, Cat# A11058, RRID: AB_2534105). Retinas were rinsed (6 × 10 minutes) in 0.1M PBS before being mounted on a on a glass slide with Aquamount and covered with a 1.5 μm thick coverslip.

Cultures were fixed in 4% paraformaldehyde (PFA), and after PBS rinse, non-specific binding was inhibited by incubation in milk powder block solution (5% dried milk powder and 0.5% Triton X-100 in PBS) for 1–2 hours (at room temperature). Cells were incubated in primary antibodies overnight at 4°C, washed in PBS and incubated with appropriate fluorescent conjugated secondary antibodies (Invitrogen) or streptavidin conjugate (Invitrogen) for 1 hour along with DAPI to counterstain nuclei. The coverslips or sections were washed and mounted for microscopy. Primary antibodies were as follows: rabbit anti-Pax6 (1:500, PRB-278P, Covance, Princeton, NJ, USA), goat anti-Otx2-biotin (1:500, BAF1979, R&D Systems, Minneapolis, MN, USA), goat anti-Sox2 (1:100, sc17320, Santa Cruz), goat anti-Lhx2 (1:250, sc-19344, Santa Cruz), Tuj1 (1:3,000, Covance), Tbr2 (1:500, ab23345, Abcam), goat anti-GIPC1 (1:100, Santa Cruz), goat anti-Brn3 (1:500, sc-6026, Santa Cruz), rabbit anti-Recoverin (1:1000, Chemicon), Lin28 (1:100, ab46020, Abcam), chicken anti-GFP (1:250, Abcam), mouse anti-Rhodopsin (1:100), goat anti-S-Opsin (1:150, Santa Cruz), goat anti-Oct3/4 (1:200, sc-8628, Santa Cruz), rabbit Nanog (1:250, ab80892, Abcam).