PCR amplifications were performed with gene-specific antisense primers tailed with a T7 RNA polymerase binding site (see Additional file 9 for primer sequences). The resulting DNA fragments were used directly as templates for synthesizing antisense ribo-probes incorporating UTP–digoxigenin (Roche) as the label with the aid of a T7 Maxi Script kit (Ambion). For miR156 and miR529 detection, 0.02 M of a 5′ digoxigenin-labeled LNA probe complementary to the target (see Additional file 9 for primer sequences) was used. In situ hybridization experiments were carried out as described by [59 (link)]. Detection was performed using the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Slides were observed and photographed using an Evolution MP5.0 color Media Cybernetics camera in conjunction with a Leica DMRB microscope, and images were processed using Photoshop CS6. Each experiment was repeated at least twice, using at least two sample series (i.e., two paraffin blocks) each time, and the reported spatial patterns were observed in at least two repeats.
Digoxigenin utp
Manufactured by Roche
Sourced in United States
Digoxigenin-UTP is a modified nucleotide used in molecular biology applications. It serves as a labeling agent for the detection and identification of nucleic acids, such as RNA and DNA, through techniques like in situ hybridization and Northern blotting.
Automatically generated - may contain errors
Lab products found in correlation
Vector blue alkaline phosphatase substrate kit 3, by Vector Laboratories (2 mentions)
Maxiscript t7 kit, by Thermo Fisher Scientific (2 mentions)
Dmrb microscope, by Leica camera (2 mentions)
Zen lite software, by Zeiss (2 mentions)
Bm purple substrate for alkaline phosphatase, by Roche (2 mentions)
Axio zoom v16 microscope, by Zeiss (2 mentions)
P6148, by Merck Group (2 mentions)
Anti fluorescein ap, by Merck Group (2 mentions)
Anti digoxigenin ap, by Merck Group (2 mentions)
Paraplast plus, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Msx2 cdna plasmid, by American Type Culture Collection (2 mentions)
Sp6 rna polymerase, by Roche (2 mentions)
Stereoscope fluorescence microscope smz1500, by Nikon (1 mentions)
Bm purple ap, by Roche (1 mentions)
Max efficiency dh5a competent cells, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Tcs sp5 microscope system, by Leica camera (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
41 protocols using digoxigenin utp
1
In-situ Hybridization for miRNA Detection
2
Visualizing Developmental Gene Expression
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RNA antisense probes were labeled with UTP-digoxigenin (11209256910, Roche Applied Science, Indianapolis, IN, United States) and used for whole-mount in situ hybridization (ISH) as described previously (Merino et al., 1998 (link)). Samples were treated with 60 μg/ml proteinase K for 25 min at 21°C for Bmp7, Fgfr1, Fgfr2, Fgfr3, Mkp3, Msx2, and Wif. Bambi required 70 μg/ml proteinase K for 28 min at 25°C; 60 μg/ml was used for 22 min at 21°C for Bmp4 and Dkk. Fgf8 was treated with 15 μg/ml for 20 min at 21°C. The hybridization temperature was 68°C, and post-hybridization washes were at 70°C for all genes. The signal of ISH was visualized with BM-Purple substrate for alkaline phosphatase (Roche Applied Science). Images were acquired with the Nikon Stereoscope Fluorescence Microscope SMZ1500 (Nikon Corporation, NY, United States) or in AxioZoom V.16 microscope (Carl Zeiss, Oberkochen, Germany) using Zen lite software (Carl Zeiss, Oberkochen, Germany).
3
In Situ Hybridization for MicroRNA Detection
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PCR amplifications were performed with gene-specific antisense primers tailed with a T7 RNA polymerase binding site (see Additional file 1 : Table S5 for primer sequences). The resulting DNA fragments were used directly as templates for synthesizing antisense ribo-probes incorporating UTP–digoxigenin (Roche) as the label in conjunction with a T7 Maxi Script kit (Ambion). For miR2118, PH12 precursor and phasiPH12-1 detection, 0.02 μM of a 5′ digoxigenin–labeled LNA probe complementary to the target (see Additional file 1 : Table S3 for primer sequences) was used. In situ hybridization experiments were carried out as described by Adam et al. (2011 (link)). Detection was performed using the Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Slides were observed and photographed by Evolution MP5.0 colour Media Cybernetics camera in conjunction with a Leica DMRB microscope and images were processed using Photoshop CS6.
4
Chick Limb Axin2 and N-Myc Expression
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The specific primers used to amplify Axin2 (403bp) were (accession number NM_204491.1): Forward-TCGAGAACAACAGCATCGTC, Reverse-GACCTGTACCCGTTCTC-CAA. N-Myc (481bp) (accession number NM_001030952.1) Forward-AGC GAC TCG GAA GAA GAA CA, Reverse-CGT CCG ATT GGA TAG ACA GAA. Briefly, RNA was obtained from chick limbs at 28 HH stage. Single-strand cDNA was synthesized with RNase H-free reverse transcriptase kit (Invitrogen, Carlsbad, USA). The P-GEM T-easy vector (Promega, Madison, USA) was used to clone fragments, and clones were obtained using MAX efficiency DH5a™ competent cells (Invitrogen). For whole-mount in situ hybridization, RNA probes were labeled with UTP-digoxigenin (Roche) as described previously (Gañan et al., 1998) . Samples were treated with 28 mg/mL proteinase K (pK) for 28 min at 20°C for all probes. They were stained with BM Purple AP (cat. 11442074001 Roche, Switzerland) and stored at 4% paraformaldehyde.
5
Whole-mount in situ Hybridization Protocol
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Whole-mount in situ hybridization was performed as previously described (deCarvalho et al., 2014 (link); Gamse et al., 2002 (link)). In brief, larvae and dissected brains were fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich) in 1 X PBS (phosphate-buffered saline) at 4 °C overnight. To synthesize RNA probes, the following restriction enzymes and RNA polymerases were used: lratd2a (BamHI/T7), fos (NotI/SP6), slc5a7a (NotI/SP6), kctd12.1 (EcoRI/T7) (deCarvalho et al., 2013 (link); Hong et al., 2013 (link)). Probes were labeled with UTP-digoxigenin (11093274910, Roche) and samples incubated at 70 °C in hybridization solution containing 50% formamide. Hybridized probes were detected using alkaline phosphatase-conjugated antibodies (Anti-Digoxigenin-AP, #11093274910, and Anti-Fluorescein-AP, #11426338910, Sigma-Aldrich) and visualized by staining with 4-nitro blue tetrazolium (NBT, #11383213001, Roche), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, #11383221001, Roche) and 2-(4-Iodophenyl)–3-(4-nitrophenyl)–5-phenyltetrazolium Chloride (INT, #I00671G, Fisher Scientific).
6
In Situ Hybridization of RNA Probes
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RNA probes were labeled with UTP-digoxigenin (Roche Applied Science, Indianapolis, IN, United States) for their use in micromass in situ hybridization, as previously described by Chimal-Monroy et al. (2002) (link). Samples were treated with 10 μg/mL of proteinase K (PK) for 5 min at 20°C for all genes. The hybridization and post-hybridization washes were at 65°C. Signal was visualized with a BM-Purple substrate for alkaline phosphatase (Roche Applied Science). Images were acquired in AxioZoom V.16 microscope (Carl Zeiss, Oberkochen, Germany) using Zen lite software (Carl Zeiss, Oberkochen, Germany).
7
Whole-mount in situ Hybridization Protocol
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Whole-mount in situ hybridization was performed as previously described (deCarvalho et al., 2014; Gamse et al., 2002) (link). In brief, larvae and dissected brains were fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich) in 1X PBS (phosphate-buffered saline) at 4 °C overnight. To synthesize RNA probes, the following restriction enzymes and RNA polymerases were used: lratd2a
(BamHI/T7), fos (NotI/SP6), slc5a7a (NotI/SP6), kctd12.1 (EcoRI/T7) (deCarvalho et al., 2013; Hong et al., 2013) (link). Probes were labeled with UTP-digoxigenin (11093274910, Roche) and samples incubated at 70 °C in hybridization solution containing 50% formamide. Hybridized probes were detected using alkaline phosphatase-conjugated antibodies (Anti-Digoxigenin-AP, #11093274910, and Anti-Fluorescein-AP, #11426338910, Sigma-Aldrich) and visualized by staining with 4-nitro blue tetrazolium (NBT, #11383213001, Roche), 5-bromo-4-chloro-3-indolylphosphate (BCIP, #11383221001, Roche) and 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5phenyltetrazolium Chloride (INT, #I00671G, Fisher Scientific).
(BamHI/T7), fos (NotI/SP6), slc5a7a (NotI/SP6), kctd12.1 (EcoRI/T7) (deCarvalho et al., 2013; Hong et al., 2013) (link). Probes were labeled with UTP-digoxigenin (11093274910, Roche) and samples incubated at 70 °C in hybridization solution containing 50% formamide. Hybridized probes were detected using alkaline phosphatase-conjugated antibodies (Anti-Digoxigenin-AP, #11093274910, and Anti-Fluorescein-AP, #11426338910, Sigma-Aldrich) and visualized by staining with 4-nitro blue tetrazolium (NBT, #11383213001, Roche), 5-bromo-4-chloro-3-indolylphosphate (BCIP, #11383221001, Roche) and 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5phenyltetrazolium Chloride (INT, #I00671G, Fisher Scientific).
8
RNA Probe Immunohistochemistry and FISH
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Immunohistochemical and fluorescence in situ hybridization was done as described (Prazak et al., 2010 (link)). Antisense RNA probes were produced using T7 or T3 RNA polymerase and digoxigenin-UTP (Roche) or fluorescein isothiocyanate (FITC)–UTP (Roche). Antibodies for fluorescent in situ mouse anti-DIG, rabbit anti-FITC, goat anti-mouse Alexa Fluor 555, donkey anti-rabbit Alexa Fluor 647, and donkey anti-goat Alexa Fluor 555 were obtained from Molecular Probes. Confocal images were acquired on a Leica TCS SP5 Microscope system.
9
In Situ Hybridization of Maize Transcripts
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Immature B73 ears (2–3 mm) were fixed in a 4% PFA solution (4g of paraformaldehyde (Sigma-Aldrich) dissolved in 100 mL of 1× PBS, pH 6.5–7), embedded in paraplast plus (Sigma-Aldrich), and sectioned to a thickness of 8 μm. To construct sense and antisense RNA probes for UB2, UB3, OBF1 and OBF4, probe fragments were amplified and cloned into pSPT18 (Roche), digested with HindIII and EcoRI, transcribed using SP6 and T7 RNA polymerase (Roche) in vitro, and labeled with digoxigenin-UTP (Roche). RNA hybridization, immunologic detection and signal capture of the hybridized probes were performed as described previously [51 ]. The in situ primers for UB2, UB3, OBF1 and OBF4 are listed in S3 Table .
10
In situ hybridization and immunocytochemistry
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