Substrates

For section immunostaining and in situ hybridization, fixed skin tissue was embedded in paraffin and sectioned at 6–7 μm. After de-paraffination, sections were processed for immunohistochemistry or in situ hybridization. The MITF antibody was from Abcam (ab12039, 1:200 dilution). The peroxidase staining was used after primary antibody treatment as described (Jiang & Chuong, 1992 (link)). Non-radioactive in situ hybridization was performed as described (Chuong et al., 1996 (link)). Briefly, the sections were treated with proteinase K (10 μg/ml in PBS) for 20 min, re-fixed with 0.2% glutaraldehyde/4% paraformaldehyde, and rinsed with PBT. The sections were then prehybridized in hybridization buffer (containing 50% formamide, 5× sodium citrate/sodium chloride buffer, 1% sodium dodecyl sulfate, 50 μg/ml heparin, 50 μg/ml tRNA) at 65°C for 1 hr. After prehybridization, sections were placed in new prehybridization buffer containing 1–3 μg/ml digoxigenin-labeled riboprobes and hybridized overnight at 65°C. Finally, sections were incubated with alkaline phosphatase-conjugated anti-digoxigenin Fab (Roche, Indianapolis, IN) overnight. Positive signals were detected by incubating the specimens with NBT (nitro-blue tetrazolium)/BCIP (5-b romo-4-chloro-3′-indolyphosphate) substrates (Promega, Madison).