Whole-mount In Situ Hybridization and TUNEL Assay

Whole-mount colorimetric and fluorescent in situ hybridizations were performed using a detailed protocol as previously described (King and Newmark, 2013 (link); Pearson et al., 2009 (link)). Fluorescence-labeled animals were mounted in ScaleA2 solution (Hama et al., 2011 (link)). Immunostaining with anti-H3P (1:1000, Millipore, Billerica, MA) was performed following fluorescent in situ development and was detected using Alexa-conjugated secondary antibody (1:1000, Abcam, Cambridge, MA). Animals were fixed and stained for TUNEL using a method previously described (Pellettieri et al., 2010 (link)) with modifications: animals were bleached in 0.075% ammonia and 3% hydrogen peroxide and treated with ProteinaseK (2 μg/ml) in PBSTx (0.3% Triton) for 10 min followed by 4% formaldehyde incubation for 10 min prior to TdT reaction.