Digoxigenin 11 utp

In situ hybridization was performed as described previously [67] (link). Antisense and sense probes were synthesized with digoxigenin-11-UTP (Roche Diagnostics) using T7 and T3 RNA polymerases, respectively. The primers used to amplify the DNA template for the probe synthesis were as follows: ADP1-INSITU-F (5′-ATG TGT AAC CCA TCA ACA ACA-3′) and ADP1-INSITU-R (5′-CGG TTA TGTTAG CAA AGG CAA T-3′).

The templates for RNA probes synthesis were amplified by PCR using primers containing T3 or T7 promoter sequence, Anti_RNA-F/Anti_RNA-R for the antisense probe and Sen_RNA-F/Sen_RNA-R for the sense probe. The antisense and sense probes were transcribed from the corresponding template using T7 and T3 RNA polymerase, respectively. Digoxigenin-11-UTP from Roche (Basel, Switzerland) was incorporated in the transcription reaction to label the probes. Whole-mount in-situ hybridization assays of zebrafish embryos and larvae were performed following a previously described protocol [67 (link)].

For fluorescent whole mount in situ hybridization (FISH), we followed the protocol outlined in (69 (link)). Triple FISH was performed as described in (70 (link)). Signal was developed with fluorophore-conjugated tyramide (1:400 reagent diluents, Perkin Elmer). Labeled probes were transcribed from linearized DNA using digoxigenin-11-UTP, fluorescein-12-UTP (Roche, Indianapolis, IN, USA), or labeled with DNP (Mirus, Madison, WI, USA) following kit instructions. SpLox, SpBrn1/2/4, SpSoxC, SpPtf1a, and SpMist probes were made as previously published [SpLox (71 (link)), SpBrn1/2/4 (25 (link)), SpSoxC (69 (link)) SpPtf1a, and SpMist (53 (link))]. SpIsl, SpNgn and SpNeuroD probes were synthetized using the following primers: SpIsl-F: 5′-CGTGGACCAGACAGACTTGA-3′; SpIsl-R: 5′-AGTCGCTGAGTGCTTTCCAT-3′; SpNgn-F: 5′-TACGACAATGATGCCCAAGA-3′; SpNgn-R: 5′-CCGTTTCACAAAGCCATTTT-3′; SpNeuroD-F: 5′-CTCGCCACCTGATCTCTAC-3′; SpNeuroD-R: 5′-TTCCCGCCTTTCAAAATATG-3′. SpANP2 probe was made as published in Woods et al. 2018. Templates of all the probes were sequenced prior to probe generation and cloned in the pGEM®-T Easy Vector (Promega, Madison, WI, USA). Samples were imaged with a Zeiss 510 Meta confocal microscope.

Fragments of each Hox gene were amplified from cDNA libraries from mixed larval and adult tissues using gene-specific primers (provided in Additional file 1: Table S2) designed in MacVector 11.0.4 based on the sequences found in the transcriptome. PCR products were cloned into pGEM-T Easy vectors (Promega, USA) and then transformed into competent Escherichia coli cells. Plasmid DNA was isolated and sequenced in both forward and reverse directions using T7 and SP6 primers. Labeled antisense RNA probes were transcribed from linearized DNA using digoxigenin-11-UTP (Roche, USA) according to the manufacturer’s instructions.

Animals were manually collected, fixed, and processed for in situ hybridization as described [79 (link),80 (link)]. Labeled antisense RNA probes were transcribed from linearized DNA using digoxigenin-11-UTP (Roche, Basel, Switzerland) or labeled with DNP (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. For I. pulchra and M. stichopi, colorimetric WMISH was performed according to the protocol outlined in [79 (link)]. For N. vectensis, we followed the protocol described by [81 ]. Double fluorescent in situ hybridization (FISH) was performed as the colorimetric WMISH with the following modifications: after the posthybridization steps, animals were incubated overnight with peroxidase-conjugated antibodies at 4 °C (anti-DIG-POD [Roche, Basel, Switzerland], 1:500 dilution, and anti-DNP [Perkin Elmer, Waltham, MA, USA], 1:200 dilution) followed by the amplification of the signal with fluorophore-conjugated tyramides (1:100 TSA reagent diluents [Perkin Elmer, Waltham, MA, USA] TSA Plus Cy3 or Cy5 Kit). Residual enzyme activity was inhibited via 45-minute incubation in 0.1% hydrogen peroxide in PTW followed by four PTW washes prior to addition and development of the second peroxidase-conjugated antibody [82 (link)].