Nbt bcip staining solution

Expression pattern of the hyLMN gene was detected in the whole mount Hydra preparations by in situ hybridization with an anti-sense digoxigenin (DIG) -labeled RNA probe [68 (link)]. DIG-labeled sense-probe was used as a control. Signal was developed using anti-DIG antibodies conjugated to alkaline phosphatase (1:2000, Roche) and NBT/BCIP staining solution (Roche). Images of the in situ preparations were collected on a Zeiss Axioscope microscope with Axiocam camera.

WISH probes were generated using the following primers: mfap4-ISH forward: 5′-TGTTCTTGGCGACGCTTCT-3′ and mfap4-ISH reverse: 5′-TAATACGACTCACTATAGGGTGATGGGTGGCATCTTCTC-3′; mpx-ISH forward: 5′-ATTAACCCTCACTAAAGGGAGTATCGAACTGCCAGCGGTGTCT-3′ and mpx-ISH reverse: 5′-TAATACGACTCACTATAGGG ACGGTCTCCTCTCTGTAGGCTCA-3′; hif-1aa-ISH forward: 5′-TCAGAGAAATGCTGGCACAC-3′ and hif-1aa-ISH reverse: 5′-TAATACGACTCACTATAGAACCCACTCCCTGTGTCTTG-3′; hif-1ab-ISH forward: 5′-CCAGTGGAACCAGACATCAG-3′ and hif-1ab-ISH reverse: 5′-TAATACGACTCACTATAGGACTTGGTCCAGAGCACGC-3′. T7 was used for transcription and digoxigenin labelling of probes. For whole-mount in situ hybridization, embryos were fixed in 4% paraformaldehyde overnight at 4 °C and subsequently dehydrated in methanol and stored at −20 °C until needed. In the first day, embryos were rehydrated to PBS/0.1% Tween-20 and then digested in 10 μg ml⁻¹ proteinase K (Roche) followed by fixation in 4% PFA. Embryos were pre-incubated with hybridization buffer at 70 °C for 3 h and then incubated with DIG-labelled RNA antisense probes at 70 °C overnight. The next day, after washing, the embryos were incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) at 4 °C overnight. The last day, after washing, the signal was visualized with NBT-BCIP staining solution (Roche)68 (link).

Specific fragments from cDNA of each neuropeptide genes were amplified using the primers with T7 or Sp6 promoter sequence at their 5′ ends (Supplementary Table S1). DIG-labeled RNA probes were prepared using the DIG RNA Labeling Kit SP6/T7 (Roche, Basel, Switzerland) with the PCR products as templates. WISH was carried out with the following protocols: the larvae were rehydrated with PBT (PBS + 0.1% Tween-20), and treated with 200 ng/μl proteinase K for 30 min to optimize hybridization; pre-hybridization was carried out for 6 h at 60 °C, then the probe was added and incubated at last 16 h at 60 °C; after the excess probe was removed by several rinses in hybridization buffer (50% formamide, 5 × SSC, 0.1% Tween, 9.2 mM citric acid for adjustment to pH 6.0, 50 μg/mL heparin, 500 μg/mL yeast RNA), the non-specific binding sites in the larval cells were blocked using the blocking buffer; the samples were incubated with the Anti-DIG-AP antibody (Roche, Basel, Switzerland) for 16 h at 4 °C; finally, the samples were stained in an NBT/BCIP staining solution (Roche, Basel, Switzerland) and kept in the dark for 1 h, and then dehydrated. The results were observed and photographed using a Nikon E80i microscope (Nikon, Tokyo, Japan). Drawings and final panels were designed using Adobe Photoshop (Adobe, San Jose, CA, USA).