In situ hybridization

Paraffin sections were deparaffinized and rehydrated, and treated with proteinase K (20 µg/ml, Invitrogen) for 10 min followed by acetylation with triethanolamine for 10 min at room temperature. After prehybridization, DIG-labeled probes (300 ng/ml) were hybridized at 65°C overnight. For the Fst in situ probe see Table S2. After washing once with 2×SSC for 20 min and four times for 20 min each with 0.2×SSC at 65°C, slides were blocked with 10% heat-inactivated sheep serum in TBS (50 mM Tris-HCl, 50 mM NaCl pH 7.5) for 1 h, and incubated with HRP-conjugated sheep anti-DIG antibody (1:1000; Roche Applied Science, 11207741910) in 1% heat-inactivated sheep serum/PBS at 4°C overnight. To detect Krt5, slides were incubated with anti-Krt5 antibody followed by secondary antibody and DAPI for counterstaining. Slides were incubated with TSA-Cy3 (PerkinElmer) for 10 min.