Dig 11 utp

Following fixation in 3.7% PFA:FSW overnight at 4°C, juveniles were washed in PBS, dehydrated through a methanol series to 100% methanol, and stored at −20°C for up to 4 weeks. A DIG‐labeled riboprobe for CapI‐vasa (Dill & Seaver, 2008) (BK006523) was generated with the SP6 MEGAscript kit (Ambion Inc., Austin, TX, USA) and DIG‐11‐UTP (Sigma 11209256910). The CapI‐vasa probe length was 1122 bp, and was diluted to a final concentration of 1 ng/μL. A DIG‐labeled riboprobe for Ct‐myc (MF693912) was generated with the T7 MEGAscript kit (Ambion) and DIG‐11‐UTP (Sigma). The Ct‐myc probe length was 964 bp, and was diluted to a final concentration of 1 ng/μL. Whole‐mount in situ hybridization was performed following published protocols (Seaver & Kaneshige, 2006). Following hybridization at 65°C for 48–72 h, the probe was detected using nitroblue tetrazolium chloride/5‐bromo‐4‐chloro‐3‐indolyl phosphate (NBT/BCIP) color substrate. Typically, the reaction was allowed to develop for 4−5 h. Extended development of the substrate reaction did not result in different expression domains for either gene. If a combination of EdU incorporation and in situ hybridization was to be performed, the in situ hybridization procedure was completed before the EdU detection reaction.

Following fixation in 3.7% paraformaldehyde in FSW overnight at 4°C, larvae were washed in phosphate-buffered saline (PBS), dehydrated through a methanol series to 100% methanol, and stored at −20°C for up to four weeks. Digoxigenin-labelled riboprobes were generated with either the SP6 or T7 MEGAscript kit (Ambion, Inc., Austin, TX, USA) and DIG-11-UTP (Sigma 11209256910). The following riboprobes and working concentrations were used: Ct-r-opsin1, 1047 bp at 0.2 ng µl⁻¹ (SP6 RNA polymerase); Ct-n-opsin1, 865 bp at 1–3 ng µl⁻¹ (T7 RNA polymerase); Ct-r-opsin3, 1176 bp at 1 ng µl⁻¹ (T7); Ct-n-opsin3, 639 bp at 1 ng µl⁻¹ (T7); Ct-n-opsin2, 722 bp at 1 ng µl⁻¹ (SP6) and Ct-r-opsin2, 620 bp at 3 ng µl⁻¹ (T7). Whole-mount in situ hybridization was performed following published protocols [42 (link)]. Following hybridization at 65°C for 48–72 h, probes were detected using nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyphosphate colour substrate. The reaction was allowed to develop for 30 min −12 h depending upon the probe. Ct-n-opsin1 was not detectable at any stages examined with 1, 2 or 3 ng µl⁻¹ of probe, multiple independent repetitions, or following resynthesis of riboprobe.

Whole-mount in situ hybridizations were performed as described elsewhere (Pearson et al., 2009 (link)). Hybridized RNA probes were labeled with DIG-11-UTP (Sigma), Fluorescein-12-UTP (Sigma) or DNP-11-UTP (Perkin Elmer) and purified as described (Lapan and Reddien, 2011 (link)). Tyramide was generated by conjugation of succinimidyl esters of rhodamine, FITC, and AMCA with tyramide-HCL (Sigma) (Hopman et al., 1998 (link)). For horseradish peroxidase enzyme inactivation, animals were incubated in 154 mmol/L sodium azide for 2 h (King and Newmark, 2013 (link); van Wolfswinkel et al., 2014 (link)). Animals were counterstained with DAPI (Sigma, 3 μg/mL in PBSTx) for 1 h and mounted for imaging.