We have previously demonstrated that a great degree of inter- as well as intra-uterus variability occurs in the staging of mouse neurulation. A high-throughput analysis using in vivo-derived samples adds a great many confounding factors into high throughput analysis. In this study, we used an established embryo culture system with a narrow mating window and matching somite numbers. Two-month-old C57BL/6 (B6) mice (weighing approximately 20 g) were purchased from Harlan, Inc., (Indianapolis, IN). Upon arrival, mouse breeders were individually housed and acclimated for at least 1 week prior to mating. Mice were maintained on a 12-hr light-dark cycle (light on: 19:00-7:00) and provided laboratory chow and water ad libitum. When a vaginal plug was detected after the mating period, it was designated as gestational day 0 (GD0) or embryonic day 0 (E0). On E8.25, dams were killed by overdose using CO2 gas. The gravid uterus was removed. Decidual tissues and the Reichert membrane were removed carefully and immediately immersed in the PBS containing 4% fetal bovine serum (Sigma, St. Louis, MO), leaving the visceral yolk sac and a small piece
