Synthesis and Application of DIG-Labeled RNA Probes

Digoxigenin (DIG)-labeled sense and antisense RNA probes were synthesized from plasmids linearized with restriction enzymes using the DIG RNA Labelling Kit (SP6/T7) (Sigma-Aldrich, St. Louis, MO). After being treated with DNase and EDTA, probes were precipitated with ethanol, dissolved in water, aliquoted, and stored at − 80°C until use. Sections were fixed in 4% (w/v) paraformaldehyde in 0.1 M PB for 10 min at RT, treated with 40 µg/mL proteinase K for 15 min at 37°C, and immersed in 0.1% (v/v) acetic anhydrate in the acetylation buffer for 15 min at RT. Hybridization was performed in the hybridization buffer ISHR7 (Nippon Gene) overnight at 55°C. Post-hybridization washing was performed in formamide/2 × saline-sodium citrate (SSC) for 1 h and 0.1 × SSC for 2 h at 55°C. The sections were incubated with anti-DIG antibody coupled to alkaline phosphatase (Roche Diagnostics, Basel, Switzerland) for 2 h at RT, and color was developed using NBT/BCIP stock solution (Roche) for signal detection.