Labeling Riboprobes for In Situ Hybridization

Single-stranded antisense riboprobes were synthesized with digoxigenin (Dig-11-UTP) (Sigma-Aldrich, St. Louis, MO), fluorescein isothiocyanate (FITC-12-UTP) (Roche, Basel, Switzerland), or 2,4-dinitrophenol (DNP) (PerkinElmer, Inc., Waltham, MA) using standard molecular methods (Collins et al. 2010 (link)). Riboprobes were detected using anti-Dig-POD (1:1000; Sigma-Aldrich, St. Louis, MO), anti-FITC-POD (1:1000; Sigma-Aldrich, St. Louis, MO), or anti-DNP-HRP (1:3000; Vector Laboratories, Newark, CA). Tyramide conjugate signal amplification was performed as previously described (King and Newmark 2013 (link)). The final incubation was with DAPI (10 µg/ml) (1:1000; Thermo Fisher Scientific, Waltham, MA). Animals were mounted in VECTASHIELD (Vector Labs, Burlingame, CA) for imaging.