Planarian RNA Expression Analysis

For RNA expression analysis in whole planarians, NBT/BCIP and Fluorescent in situ hybridizations were performed as previously described (73 (link)). Following hybridization, DIG-probes were detected with antibodies in MABT containing 5% horse serum appropriate for NBT/BCIP in situ hybridization (Roche 16646822, anti-DIG-AP, 1:1000) or for FISH (Roche 28557000, anti-DIG-POD, 1:1000 or Roche 20052220, anti-FLCN-POD, 1:1000). NBT/BCIP developed animals were stored in 80% glycerol overnight prior to mounting, while FISH animals were soaked overnight in a modified ScaleA2 solution (40% glycerol, 2.5% DABCO, 4M urea) prior to mounting and imaging. Anti-phospho-histone H3 (Ser10)(H3P) antibody (1:500, abcam, ab32107) and Alexa-conjugated goat anti-rabbit secondary antibodies (1:1000, abcam, ab150086) were used to stain mitotic cells.