4oh tmx

E10.5 Sptlc2^f/fCdh5^CreERT2/+ embryos were harvested, washed once with pre-warmed PBS, placed in DMEM-F12 media (Thermo Fisher) supplemented with 5% FBS and equilibrated in 37°C incubator with 5% CO_2. The extra-embryonic tissues were removed using a dissection microscope. Embryos were divided in two groups. The experimental group was cultured in embryo culture medium containing DMEM-F12 (Thermo Fisher), 15% KSR (Thermo Fisher), 1x N2 supplement (Thermo Fisher), 1x Glutamax (Thermo Fisher), 2.5 µL/mL of 100x Pen-Strep solution (Thermo Fisher), 1 µM 4-OH-TMX (Sigma), 5 µM D-erythroSO-BSA complexes and 25 nM 1-deoxySO-BSA complexes. Control group embryos were cultured similarly but received vehicle-BSA complexes in place of 1-deoxySO-BSA complexes. Cultures were carried out in 50 mL conical tubes in high O₂ atmosphere which was created by attaching a Steriflip filter unit (Millipore Sigma) connected to an O₂ tank for 20–30 sec. The tubes were sealed with parafilm and placed in roller bottle in a 37°C incubator with 5% CO₂ for 24 hours. The embryos were then washed in PBS with Ca⁺² and Mg⁺² (Thermo Fisher) and fixed overnight in Ca⁺²Mg⁺² PBS plus 0.4% PFA (Electron Microscopy Sciences 15710). The fixed embryos were washed with PBS and stored in PBS plus 0.1% Sodium Azide. Embryos were genotyped and processed for light sheet microscopy as described above.