2.4. β-Galactosidase and alkaline phosphatase staining

For β-Gal staining on frozen sections, 0.2% glutaraldehyde/PBS was used for fixation prior to staining. Sections were then washed and stained overnight at 37 °C as previously described (Ishii et al., 2003). To count PGCs at E8.5, embryos were dissected and fixed for 1 h in 4% PFA/PBS at 4 °C, washed three times in cold PBS and treated with 70% ethanol for 1 h at 4 °C. After washing another three times with distilled water, they were stained with α-naphthyl phosphate/fast red TR for 15 min at room temperature (Ginsburg et al., 1990). Embryos were then rinsed in water and preserved in 70% glycerol. The anterior portion of each embryo was collected for genotyping, and the posterior portion was flattened under a coverslip for counting. Alkaline phosphatase staining on frozen sections was performed according to the method reported previously (Ishii et al., 2003).