Whole retina and lens were dissected away from other ocular tissues (explants) and placed into a 14 ml round bottomed snap cap tube with 1 ml of retina culture medium (RCM) and rotated at 15 RPM on a carousel with an axis of rotation at 30° above horizontal. Explants were incubated at 37 °C in a humidified, 5% CO2 atmosphere. RCM is composed of 1 x DMEM/F12 (US Biological, Cat# D9807-05), 1% fetal bovine serum (Thermo Fisher Scientific, Cat#16140071), 6 mg/ml glucose (Sigma, Cat# G7528), 0.1% NaHCO3 (Thermo Fisher Scientific, Cat#25080–094), 50 mM HEPES (Thermo Fisher Scientific, Cat# 15630–080), 1 mM glutamax (Thermo Fisher Scientific, Cat# 35050–061), 1 x N2-plus supplement (R&D Systems, Cat#AR003), and 1 x penicillin/streptomycin (Thermo Fisher Scientific, Cat# 15070–063).
At the end of the culture period, explants were rinsed in PBS. For in situ hybridization or immunohistology, explants were fixed in 4%PFA and prepared for cryostorage (see above). For qPCR, lens tissue was removed, and retinas were snap frozen in liquid N2, and stored at –80 oC until use.
At the end of the culture period, explants were rinsed in PBS. For in situ hybridization or immunohistology, explants were fixed in 4%PFA and prepared for cryostorage (see above). For qPCR, lens tissue was removed, and retinas were snap frozen in liquid N2, and stored at –80 oC until use.