Two ages of transgenic mice were studied: embryonic day (ED) 18.5 and postnatal day (PD) 30. Mice were anesthetized with pentobarbital and perfused transcardially with cold 0.1 M sodium phosphate buffer/2 mM MgCl2 followed by fixative (cold 4% paraformaldehyde). Tissues were then dissected and post-fixed for 5-6 hours (ED18.5) or 4 hours (PD30). Fixed tissues were transferred to 30% sucrose/2 mM MgCl2, in 1X phosphate-buffered saline (PBS) and incubated at 4°C overnight. Tissues were embedded in Tissue-Tek (Miles, Indiana, USA) and quick frozen on dry ice. If not used immediately, the samples were stored at −70°C. Sectioning was done on a Leica CM3050S cryostat at −30°C generating either 14 μm (ED18.5) or 25 μm (PD30) thick sections that were transferred directly onto Superfrost glass slides (Fisher, Pennsylvania, USA). Slides were air-dried at room temperature, washed with sodium phosphate buffer and 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
