Two-Color in Situ Hybridization for Mouse Brain

The first-pass single-color ISH analysis was performed essentially as described previously^{29 (link)}. The accession numbers of the DNA templates used for probe generation and the genomic sequence (mm9 assembly) that was amplified by PCR for template generation are listed in Supplementary Table 3. For the two-color section ISH analysis, maternal animals were anesthetized and embryos were immediately dissected out. The brains were removed, fixed overnight in 30% sucrose/4% paraformaldehyde (vol/vol) and sectioned in the coronal plane on a Leica sledge microtome at 40 μm. Sections were individually mounted on slides and processed for ISH to visualize expression of various genes. Single- or two-color nonradioactive ISH was performed using previously described method with some modifications^{32 (link)}. Glass slides with sections were fixed by 4% paraformaldehyde and treated with proteinase K (Roche). Sections were then fixed again and hybridized with digoxigenin-labeled or fluorescein-labeled probes (Roche) at 70 °C overnight. Excess probes were washed out and sections were blocked with lamb serum and incubated with solution containing alkaline phosphatase–conjugated with digoxigenin-labeled or fluorescein-labeled antibodies (Roche). Color was developed with combinations of the chromagens nitroblue tetrazolium (Nacalai, 350 mg ml⁻¹) and 5-bromo, 4-chloro, 3-indolylphosphate for blue staining or tetranitroblue tetrazolium (Research Organics, 350 mg ml⁻¹) and 5-bromo, 4-chloro, 3-indolylphosphate for brown staining. The probes used for this analysis that gave good signal using two-color ISH are listed in Supplementary Table 4, whereas probes that were tested, but did not give good signal, are included in Supplementary Table 3. All two-color ISH data using candidate genes and Shh can be accessed at http://blackshaw.bs.jhmi.edu and in the Mouse Gene Expression Database at Jackson Labs (http://www.informatics.jax.org/expression.shtml).