then incubated overnight at 37°C with β-gal staining solution (0.1 M NaHPO4, 0.1 M NaH2PO4, 2 mM MgCl2, 0.1% sodium deoxycholate, 0.02% NP-40, 10 mM K3(Fe)CN6, 10 mM K4(Fe)CN6, 1 mg/ml X-gal). The slides were subsequently washed with 1X PBS and incubated with distilled water either for 1 hour (ED18.5) or overnight (PD30). Slides were then counter-stained with Neutral Red (1% w/v in 37 mM sodium acetate), dehydrated through a graded series of ethanol (50%, 70%, 90% and 100%) and cleared with xylene. The slides were air-dried overnight at room temperature in a fume hood followed by the application of cover slips. Microscopy was done using a Zeiss Axiovert 200M microscope with a high resolution Retiga 1300R CCD camera and Slidebook image analysis software. Anatomical analysis was done with the aid of Kaufman (Kaufman, 1995) and Paxinos and Franklin (Paxinos and Franklin, 2001).
