LacZ Staining and Eosin Counterstaining of Embryos

LacZ staining of embryos was performed as previously described [20] (link), [21] (link), except that 2.5% GDA in PBS was used for fixing embryos upon collection. For section staining with eosin, 4 µm thick LacZ stained sections were further immersed in eosin for 2 min, rinsed in runnig water for 5 min, dehydrated serially 3×2 min in 90% ethanol, 3×2 min in 100% ethanol, 2 min in 1∶1 solution of ethanol: histochoice clearing agent (Sigma), 3×2 min in histochoice clearing agent, then mounted with Entellan (Merck), and photographed. Pictures of whole embryos were acquired using a Zeiss stereoscope equipped with camera and Axiovision software, whereas a Nikon Eclipse Ti microscope was used to photograph LacZ/eosin stained embryo sections.