1. Fix embryos in 4% paraformaldehyde, 0.2% glutaraldehyde, 4% sucrose, 0.15 mM CaCl2, 0.1 M sodium phosphate, pH 7.3, for 30 min at 4°C.
3. Incubate in 4% 5-bromo-4-chloro-3-indolyl-P-d-galactopyranoside, 150 mM NaCl, 1 mM MgCl2, 1.5 mM K4(Fe3[CN]6), 1.5 mM K3 (Fe2[CN]6) in 5 mM sodium phosphate buffer at 37°C for 3 h.
4. Rinse the embryos in 0.1 M sodium phosphate.
5. Mount in glycerol or dehydrate through alcohol series, xylene, and mount in Durcupan.
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