Mitf antibody

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).

Tyrosinase antibody (used at 1:1000) was a gift from R. Halaban, Yale University School of Medicine (85 (link)). Tyrosinase antibody (T311) was purchased from Santa Cruz Biotechnology (1:200; catalog no. sc-20035, lot no. J0616). MITF antibody was purchased from Abcam (1:500; catalog no. ab13703, lot no. GR27425–10). MITF (D5G7V; 1:1000; catalog no. 12590S, lot no. 1), phosphor-(Ser/Thr) PKA substrate (1:1000; catalog no. 9621, lot no. 14), and GAPDH (14C10;1:1000; catalog no. 2118S, lot no. 8) antibodies were purchased from Cell Signaling Technology. Antibodies directed against (R21) (3.2 mg/ml, 1:2000) were used as previously described (28 (link)).