Skin permeability assay.
Unfixed and freshly isolated E18.5 embryos were rinsed in PBS and then submerged in X-gal reaction mix at pH 4.5 (100 mM NaPO4, 1.3 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, and 1 mg/ml X-gal) at room temperature overnight [36]. At this pH in the absence of epidermal barrier, the solution penetrates epidermis and an endogenous β-galactosidase–like activity catalyzes production of a blue precipitate. After staining, embryos were washed twice in PBS, fixed overnight at 4 °C in 4% paraformaldehyde, and were photographed using a 35 mm Nikon digital camera.