Optimized FISH Protocol for Planarian

FISH protocols were performed as previously described [58 (link),59 (link)] with the following modifications. Asexual and sexual hatchling/sexual adult planarians were killed in 7.5% N-acetyl-L-cysteine in 1X PBS for 5/10 minutes; fixed in 4% formaldehyde in PBSTx (1X PBS + 0.1% Triton X-100) for 15/30 minutes; bleached in Bleaching Solution (1X SSC solution containing 5% deionized formamide and 1.2% hydrogen peroxide) for 2/4 hours; incubated in PBSTx containing 10 μg/ml Proteinase K and 0.1% SDS for 10/20 minutes; and refixed in 4% formaldehyde in PBSTx for 10/15 minutes. Planarians were blocked in Blocking Solution (5% heat inactivated horse serum, 5% Roche Western Blocking Buffer in TNTx [0.1 M Tris pH 7.5, 0.15 M NaCl, 0.3% Triton X-100]) for 2 hours at room temperature, and incubated in Blocking Solution containing anti-Digoxigenin-POD (1:2,000), anti-Fluorescein-POD (1:2,000), or anti- Dinitrophenyl-HRP (1:2,000) for 8 hours at 12°C. For fluorescent development of riboprobes, TSA reactions were performed for 30 minutes.