Digoxigenin labeled utp

DNA templates were generated by RT-PCR. The Arc DNA template was designed to amplify a fragment of the mouse Arc gene from bases 1699–2851 (NCBI RefSeq: NC_000081.7, provided by T. Kitsukawa, Ritsumeikan University, see Supplementary Fig. 9). The Fos DNA template was designed to amplify a fragment containing an intron sequence of the mouse Fos gene from bases 292–1061 (NC_000078.7, provided by T. Kitsukawa, Ritsumeikan University). The Homer1a (H1a) DNA template was designed to amplify a fragment on the 3’-untranslated region of transcript variant S of the mouse H1a gene from bases 52,325–53,088 (NC_000079.7, provided by K. Akama, Rockefeller University). Antisense RNA probes were synthesized with a mixture of either digoxigenin-labeled UTP (Roche Diagnostics) or fluorescein-labeled UTP (Roche Diagnostics) and purified using Probe quant G-50 Micro columns (GE Healthcare).

To synthesize anti-sense RNA probe for whole-mount in situ hybridization, cDNA was PCR-amplified from total RNA extracted at 24 hpf embryos. The PCR fragments were cloned into the pGEM®-T Easy Vector (Promega, Madison, WI, United States). The cloned vectors were linearized with the NcoI restriction enzyme and then transcribed in vitro using SP6 RNA polymerase (Thermo Scientific, Waltham, MA, United States) with digoxigenin-labeled UTP (Roche, Basel, Switzerland). The following primers were used: odf3b (NCBI, acc. no. NM_199958) forward 5′-AGA​CGT​CAT​GTC​ACC​TGT​G-3′ and odf3b reverse 5′-ATG​ACC​TGC​TTA​ATC​TTC​ACC​ATC​C-3′. Whole-mount in situ hybridization was performed as described previously (Thisse and Thisse, 2008 (link)).

In situ hybridization experiments followed standard procedures. Samples fixed in 4% paraformaldehyde (PFA) were dehydrated by passage through a graded ethanol series and embedded in paraffin. Serial tissue sections were treated with proteinase K for 15 min at room temperature. TRPM7 riboprobes were generated by in vitro transcription using digoxigenin-labeled UTP, in accordance with the manufacturer’s instructions (Roche Diagnostics Corp., Indianapolis, IN, USA). Several negative controls (e.g. sense probe and no probe) were run in parallel with the experimental reaction.

Sense and Antisense probes were in vitro transcribed using T7 RNA polymerase (New England Biolabs, Ipswich, AU) in the presence of digoxigenin-labeled UTP (Roche, Basel, CH) from PCR amplified templates that included a T7 promoter. Tissues were fixed in 4% paraformaldehyde, paraffin embedded and sectioned at 8 μm. Sections were mounted on Probe-On-Plus slides (Fisher Scientific, Pittsburgh, US). Prehybridization, hybridization and detection were essentially following Carr & Irish [55 (link)].