Inflorescences were fixed in FAA buffer containing 3.7% (v/v) formaldehyde and vacuum-infiltrated for 15 min on ice. Samples were dehydrated in a graded ethanol series and stained with safranine in xylene/ethanol solutions. Samples were placed into a 60 °C oven for 1 wk and finally embedded in Paraplast (Leica, Germany). Transverse sections of 8 μm in thickness were transferred onto poly-L-lysine coated glass slides (Sigma-Aldrich, USA) for hybridization. RNA in situ hybridization was performed using a Digoxigenin RNA Labeling Kit (Roche, USA). A 497-bp SKS18 cDNA fragment and a 449-bp VTC1 cDNA were amplified and cloned into the pBluescriptSK vector. These plasmids were individually digested by BamHI or EcoRI and used as templates. Sense and antisense probes were transcribed using the above templates by the T3 or T7 RNA polymerase (Roche, USA), respectively. The hybridization for SKS18 transcripts in WT and tdf1 anthers was performed in a single batch, while that in ams was performed in another batch. The hybridization for VTC1 transcripts in WT and tdf1 anthers was performed in a single batch. Primer sequences are provided in Supplemental Data Set S1 .
Digoxigenin rna labeling kit
Manufactured by Roche
Sourced in Switzerland, Germany, United States, China
The Digoxigenin RNA Labeling Kit is a laboratory tool used for the incorporation of digoxigenin-labeled nucleotides into RNA molecules during in vitro transcription. This kit provides the necessary reagents to label RNA probes that can be used in various applications, such as Northern blotting, in situ hybridization, and RNA protection assays.
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Lab products found in correlation
Pgem t easy vector, by Promega (4 mentions)
Pgem t vector, by Promega (4 mentions)
Pgem t plasmid, by Promega (4 mentions)
Levamisole, by Merck Group (4 mentions)
Alkaline phosphatase labeled anti digoxigenin and bm purple, by Roche (3 mentions)
Sheep anti digoxigenin antibody conjugated to alkaline phosphatase, by Roche (3 mentions)
Paraplast plus, by Merck Group (2 mentions)
Paraplast, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Nitro blue tetrazolium, by Avantor (2 mentions)
5 bromo 4 chloro 3 indolyl phosphate, by Avantor (2 mentions)
Pgem t easy, by Promega (2 mentions)
3 aminopropyltriethoxysilane, by Merck Group (2 mentions)
Paraplast, by Leica (1 mentions)
T7 rna polymerase, by Roche (1 mentions)
Poly l lysine coated glass slide, by Merck Group (1 mentions)
Sigmaspin sequencing reaction clean up, by Merck Group (1 mentions)
Peasy t3, by Transgene (1 mentions)
Microtome, by Leica (1 mentions)
Eclipse ci, by Nikon (1 mentions)
58 protocols using digoxigenin rna labeling kit
1
In Situ Hybridization of Anther Transcripts
2
Whole-mount in situ Hybridization Protocol
3
In situ Hybridization of SCP46 Gene
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Nipponbare seeds at different developmental stages were fixed in diethyl pyrocarbonate (DEPC)-treated 70% FAA fix solution (70% ethanol, 5% glacial acetic acid and 3.7% formaldehyde) overnight at 4°C, then dehydrated through a series of ethanol solutions, infiltrated with xylene and embedded in paraffin. The embedded tissues were cut into 9-μm-thick sections. The SCP46 probe was PCR amplified by primer set In-SCP46F+In-SCP46R. After being cloned into pGEM-T Easy vector (Promega, Madison, U.S.A.), the segmental sequence was transcribed in vitro to synthesize sense and antisense probes from either T7 or SP6 promoter using the Digoxigenin RNA labeling kit (Roche, Basilea, Swiss Confederation). The sense strand was used as a negative reference. The hybridization and immunological detection were conducted as described by Zhang et al.2010 [59 (link)].
4
Zebrafish Gonadal Development Analysis
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Gonads were isolated from the zebrafish developed from MTZ-treated embryos or control embryos without MTZ treatment at 20 dpf and 100 dpf under the stereoscope. They were fixed in 4% paraformaldehyde solution overnight at 4 °C, dehydrated and embedded as described above, then sectioned at 7 μm transversely. Some sections stained with H&E and others for hybridizations. A 1223 bp vasa cDNA fragment was inserted into a pGEM-T vector for the synthesis of antisense RNA probes under the drive of T7 promoter by using the digoxigenin RNA Labeling Kit (Roche). The RNA probes were treated with RNase-free TURBO DNase and purified with SigmaSpinTM Sequencing Reaction Clean-Up (Sigma). The sections were digested with proteinase K (10 μg/ml) for 10 min and hybridized with the probes at 65 °C for 14 hours. Chemical in situ hybridization was conducted by developing the signals with BCIP/NBT substrates on sections and post fixed in 50% glycerin. The results was photopgraphed under microsope uisng digital CCD camera.
5
Transcriptional Regulation of Zebrafish Skeletal Development
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Embryos were collected and fixed in fresh 4% paraformaldehyde, dehydrated in methanol and stored at -20 °C until use. In situ probe primers targeting Sp7 transcription factor (sp7) (also known as osterix), α1 chain of collagen type X (col10a1), connexin 43 (cx43), distal-less homeobox 2b (dlx2b), and even-skipped-like1 (eve1) were synthesized and listed in Table S2 . All probes were amplified from cDNA derived from 2 dpf embryos and subcloned into pEASY-T3 (TransGen Biotech, Beijing, China). In situ probe synthesis was performed using Digoxigenin RNA Labeling Kit (SP6/T7, Roche Diagnostics, Mannheim, Germany). Whole-mount in situ hybridization (WISH) was performed using the standard procedure 31 (link). Embryos were mounted into glycerin, and imaged using Leica M205 FC Stereomicroscope (Leica Microsystems Nussloch GmbH, Heidelberger, Germany).
6
Detecting Pbf1 Gene Expression via In Situ Hybridization
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The specific fragment of Pbf1 was amplified using cDNA as template, and was inserted into the pGEM-T Easy vector (Promega, Madison, WI) for sequencing. According to the method of Zhang (2015) (link), the sense probe was then generated using primers T7-PBF1-F and PBF1-R, and the antisense probe using primers PBF1-F and T7-PBF1-R Supplemental Data Set S12 ). Sense and antisense probes were transcribed in vitro from the T7 promoter with T7 RNA polymerases using the digoxigenin RNA-labeling kit (Roche, Basel, Switzerland). Fifteen DAP B73 seeds from plants grown under SN and DN conditions for in situ hybridization were fixed and embedded in Paraplast Plus (Sigma-Aldrich, St. Louis, MO, USA) embedding medium. Nonradioactive RNA in situ hybridization with digoxigenin-labeled sense and antisense probes was performed on 10-μm sections of tissues as described by Coen et al. (1990) (link).
7
Fluorescence In Situ Hybridization with Paraffin Wax
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We performed FISH with paraffin wax. The leaf veins of Micro-Tom, which were approximately 1 cm in length, were fixed for 24 h at 4 °C in freshly prepared 4% (w/v) paraformaldehyde buffered with phosphate-buffered saline66 (PBS, pH 7.2). Fixed tissues were dehydrated in a graded ethanol series and impregnated with paraplast (P3683-1kg, Sigma). They were then cut into 10-μm slices using a microtome (Leica Microsystems, Wetzlar, Germany). To generate the antisense and sense probe, the specific CDS region (408 bp) was amplified using the Digoxigenin RNA labeling kit (Roche, Switzerland). The primers used to amplify the template are listed in Supplementary Table S7 . Dewaxed slices were mixed with the two probes (2 ng/μL) and hybridized in hybridization buffer for 12 h at 55 °C. The sections were then transferred onto slides when the signal was sufficiently strong, and images were obtained using a fluorescence microscope (Nikon Eclipse Ci, Japan). The excitation/emission (Ex/Em) filters for blue and red fluorescence were 380/420 nm and 560/590 nm, respectively.
8
Synthesis of MyHC RNA Probes
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MyHC DIG-labeled RNA probes were synthesized as previously described [38 (link)]. Briefly, target sequences of mouse MyHC gene were obtained by RT-PCR using gene-specific primers (Table 1 ). The short (564 bp) and long (835 bp) RT-PCR products were cloned into the pDrive vector. For the preparation of the antisense RNA probe, the plasmids were linearized with restriction enzymes (Table 1 ) and synthesized with T7 or SP6 RNA polymerase. The sense RNA probes for MyHC were used as control. The probes were labeled with the digoxigenin-RNA-labeling kit (Roche, Germany). MyoG and Sox10 chicken probes were cloned in our laboratory [28 (link)].
9
In Situ Hybridization of CsAP3 in Flower Buds
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Shoot tips containing flower buds at various stages were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. After fixation, tissues were washed, dehydrated, and embedded in wax for sectioning and in situ hybridization as described (Zhang et al., 2013 (link)). Tissues for whole-mount in situ hybridization were not embedded, and after dehydration, the procedure was performed according to the methods previously reported (Hejatko et al., 2006 (link)). CsAP3-specific regions were amplified with corresponding primer pairs (see Supplementary Table S2 ) and transcribed in vitro as probes using the Digoxigenin RNA labeling kit (Roche # 11175025910).
10
Prothalli and Pinnae Fixation and In Situ Hybridization
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Prothalli and pinnae at various stages were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. After fixation, tissue samples were washed, dehydrated, and embedded in wax for sectioning and in situ hybridization as described by Zhang et al. (2013) (link). AcLEC1-specific regions were amplified with primer sets 5′-GAAGATAGCAGATGATGCCAAGG-3′ and 5′-ATGAATCCCCCCGATACTACTAA-3′ and transcribed in vitro as probes using the Digoxigenin RNA labeling kit (Roche, Mannhein, Germany).
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