In Situ Hybridization and Antibody Staining of Planarian Neoblasts

RNA probes were synthesized in vitro using Sp6 or T7 polymerase (Roche) and DIG-, FITC-, or DNP-modified (Perkin Elmer) nucleotides. RNA probes were purified and precipitated with ethanol and 7.5 M ammonium acetate. For ISH and fluorescent in situ hybridization (FISH), animals were fixed and processed as previously described [66 (link),67 (link)]. After probe development, neoblasts were visualized with the rabbit anti-SMEDWI-1 antibody (1:1,000; kindly provided by Kerstin Bartscherer, Max Plank Institute for Molecular Biomedicine, Münster, Germany) [47 (link)]. Nuclei were stained with DAPI (1:5,000) and mounted with 70% glycerol in PBS.