Digoxigenin rna labelling kit

To create the OsNRAMP5-promoter:GUS construct, 2.06kb of the genomic sequence located upstream of the OsNRAMP5 initiation codon was amplified by PCR from Zhonghua 11 genomic DNA using primers Pnr5-F (5’-agggatcccgcaactcccacaactactg-3’) and Pnr5-R (5’-tcggatccgcttcctctcttagcttcttca-3’). The amplified promoter fragment was digested by BamH1 and introduced into the vector pDX2181 in the correct direction (Ye et al., 2012 (link)). Wild-type Zhonghua 11 calli was transformed with this construct. The transgenic plant tissues were incubated in a X-Gluc staining buffer at 37 °C for 4h (Jefferson et al., 1987 (link)).
Hybridization and immunological detection were performed as described in Xue et al. (2008) (link). The probe of OsNRAMP5 was amplified from Zhonghua 11 by PCR using gene-specific primers in situ-F (5’-cgacgagcccttgccgta-3’) and in situ-R (5’-tctgcgagcgatctggacc-3’) and cloned into the pGEM-T vector (Promega). The sense and antisense probes were transcribed in vitro by T7 or SP6 transcriptase using a Digoxigenin RNA labelling kit (Roche).

To study mRNA expression, whole mount in situ hybridization was performed using sense and antisense riboprobes prepared with a digoxigenin RNA labelling kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. At least three mouse E9.5 embryos (25–30 somites) were analyzed per probe, as described previously (Ybot-Gonzalez et al., 2005 (link)). Selected embryos, labelled for Daam1 (Ybot-Gonzalez et al., 2007 (link)), glypican4 (Ybot-Gonzalez et al., 2005 (link)) or Wnt5a (Gavin et al., 1990 (link)) were embedded in a gelatin-sucrose-albumin and glutaraldehyde solution, and vibratome sections (50 μm) of the embryos were photographed by using an Axiophot (Zeiss, Jena, Germany) photomicroscope. To compare Daam1 expression between control and treated embryos, the in situ hybridization procedure was performed in only one tube, adequately marking the embryos for their later identification. Sense-strand control riboprobes gave no specific signal.
Embryos at E11.5 and E12.5 were fixed in 4% PFA, and they were immediately whole mount stained using the X-Gal method (Carroll et al., 2003 (link)). Eosin counterstained paraffin sections (7 μm) were examined.

Tissue preparation and in situ hybridization was performed according to a standard protocol61 (link). LM15_RMs2 anthers at different stages were harvested and fixed in FAA (10% formaldehyde:5% acetic acid:50% alcohol) for 12 h at 4 °C. Probe templates were amplified from the Ms2 cDNA using PCR primers P146 and P147. Both sense and antisense RNA probes were prepared independently using the digoxigenin RNA labelling kit (Roche Diagnostics, Mannheim, Germany).

A 478‐bp fragment of GhNSP cDNA was amplified from the cDNA, which was made from the stage 8 anther of 1355B plants, with the primers GhNSP‐S and GhNSP‐AS (Table S1). The PCR product was cloned into the pGEM‐T‐Easy vector (Promega, Madison, WI, USA) and sequenced. Sense and antisense probes were transcribed in vitro from the T7 or SP6 promoter with respective RNA polymerases using the digoxigenin RNA‐labelling kit (Roche). Tissue sections were prepared as described by Min et al. (2013 (link)). In brief, samples were fixed in FAA [10% formalin, 5% acetic acid and 50% ethanol (v/v) in RNAase‐free water]. After dehydration and embedding of the tissue in paraffin wax, the sample blocks were sectioned into 10‐μm slices using the microm HM 340E microtome (Thermo Scientific, Waltham, MA, USA) and were applied to RNAase‐free glass slides. The sections were then dewaxed, rehydrated, prehybridized, hybridized and visualized as described by manufacturer’s instructions. Hybridization was detected by using the antidigoxigenin‐alkaline phosphatase conjugate (Roche, Mannheim, Germany), visualized by incubation with NBT/BCIP stock solution (Roche), and images were captured using a Zeiss Axio Scope‐A1 microscope.

SpPou4f1/2 probes were generated by cloning full-length SpPou4f1/2 cDNA into pIRES-hrGPF-2a vector (Agilent). The Pax6 probe has been described in Ullrich-Lüter et al. [10 (link)]. Both antisense- and sense-digoxigenin-labelled SpPou4f1/2 probes were obtained using a digoxigenin-RNA labelling kit (Roche), following the manufacturer's instructions by using 1 µg of linearized plasmids. The Pax6 RNA probe was similarly prepared using unlabelled ribonucleotides and was subsequently labelled with 2, 4-dinitrophenyl using a Label-it kit (Mirus) following the manufacturer instructions. Whole-mount in situ hybridization and immunostaining against Sp-Opsin4 followed the protocol of Ullrich-Lüter et al. [10 (link)]. Two-colour in situ staining was performed as described in Cole et al. [22 (link)]. After staining, samples were mounted in glycerol and analysed on a Leica TCS SP2 confocal laser-scanning microscope.

Whole-mount in situ hybridisation was carried out using sense and antisense digoxigenin-labelled RNA probes prepared using a digoxigenin RNA labelling kit (Roche, 11175025910) according to the manufacturer's instructions. As described previously (Ybot-González et al., 2005 (link)), mouse embryos were analysed with the probe for Sox10 (Britsch et al., 2001 (link)). Whole embryos were photographed on a stereomicroscope and were embedded in a gelatine-sucrose-albumin and glutaraldehyde solution. Vibratome transverse sections of the caudal NT (50-μm-thick) of the embryos were photographed at 20× and 40× on the Olympus BX-61 microscope using the same imaging parameters for all samples. CAs were identified by the presence of dorsal Sox10 labelling.

RNA in situ hybridization was performed as described previously [28 (link), 29 (link)]. Briefly, the specific region for the selected genes was cloned into pGEM-TEasy (Promega) vector, and then Digoxigenin RNA labelling kit (Roche) was used for in vitro transcription and labelling. After hybridization and immunological detection, signals were visualized under Leica DM6 B microscopy with bright-field mode. Primers of 5′-GCTGATATCCTTGCTCTGGTAGC-3′ and 5′-TAATACGACTCACTATAGGGACCTGATCCTTTGCGTCAAGG-3′ were used to generate an antisense probe of LOC_Os04g53640, and primers 5′-TAATACGACTCACTATAGGGGCTGATATCCTTGCTCTGGTAGC-3′ and 5′-ACCTGATCCTTTGCGTCAAGG-3′ were used to generate its sense probe.