2.3  X-Gal staining
The X-Gal buffer and heterozygous embryos were warmed to 37 °C. X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; Multicell) dissolved in dimethyl-formamide (100 mg/ml) was then added to the X-Gal buffer (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide in rinse buffer) immediately before use to a final X-Gal concentration of 1 mg/ml. The samples were incubated in this X-Gal staining solution for 24 h at 37 °C in total darkness with abundant movement for equal exposure to the staining solution. The samples were thoroughly washed in the rinse buffer on ice until the solution was completely free of yellow coloration from the X-gal buffer. They were then fixed in 4% PFA for minimum 48 h. The samples were gradually dehydrated to 70% ethanol for storage and imaging. For paraffin embedding, the samples were dehydrated gradually to 100% ethanol and incubated respectively in 50/50 ethanol/xylene, 2× 100% xylene, 50/50 xylene/paraffin for maximum 5 minutes each step and were finished with 2× paraffin at 60–62 °C for one hour. The samples were sectioned at 10 μm and counterstained with Nuclear Fast Red (NFR). For cryosectioning, the samples were submerged in a 1:1 ratio of 50% sucrose:Optimal Cutting Temperature (OCT) overnight and frozen in OCT with ethanol slurry on dry ice (OCT: Leica). The samples were equilibrated to the cryotome׳s working temperature (−20 °C), then sectioned at 12 μm to be counterstained with NFR.