Cerebella were dissected from the adult control and DKO mice and snap-frozen in OCT compound (Tissue-tek). Then, 15 μm sections were cut using Leica CM1950 cryostat and briefly fixed with 4% paraformaldehyde (PFA) in diethyl pyrocarbonate-treated phosphate-buffered saline (PBS) for 10 min. After prehybridization treatment, the sections were hybridized with the digoxigenin-labelled riboprobes against Espn, synthesized using transcriptional kit (Roche) from Espn-pBluescript with T3 RNA polymerase (Promega), at 65 °C overnight. After several rounds of washing with different dilutions of SSC, the sections were blocked at room temperature for 1 h. The sections were then incubated with 1:1,000 anti-digoxigenin alkaline phosphatase-conjugate (Roche) at 4 °C overnight. The signals were visualized by staining with 250 μg ml−1 nitroblue tetrazolium and 125 μg ml−1 BCIP (5-bromo-4-chloro-3-indolyl-phosphate) at 37 °C overnight. Images of sections were captured by Olympus BX43 microscope equipped with DP72 camera.
Anti digoxigenin alkaline phosphatase conjugate
Manufactured by Roche
Sourced in Germany
The Anti-digoxigenin alkaline phosphatase conjugate is a laboratory reagent used to detect the presence of digoxigenin, a small molecule often used as a label in various biological applications. The conjugate consists of an anti-digoxigenin antibody linked to the enzyme alkaline phosphatase, which can catalyze a colorimetric or chemiluminescent reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Nbt bcip stock solution, by Roche (2 mentions)
Digoxigenin rna labelling kit, by Roche (2 mentions)
Microm hm 340e microtome, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Digoxigenin 11 utp, by Roche (2 mentions)
Axio scope a1 microscope, by Zeiss (2 mentions)
Cm1950, by Leica (1 mentions)
T3 rna polymerase, by Promega (1 mentions)
Dp72 camera, by Olympus (1 mentions)
Bx43 microscope, by Olympus (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Proteinase k, by Promega (1 mentions)
Nitroblue tetrazolium 5 bromo 4 chloro 3 indolyl phosphate, by Roche (1 mentions)
Pcr blunt 2 topo vector, by Thermo Fisher Scientific (1 mentions)
Glycerol gelatine, by Merck Group (1 mentions)
Megascript sp6 or t7 kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti digoxigenin alkaline phosphatase conjugate
1
In Situ Hybridization of Espn in Mouse Cerebellum
2
In Situ Hybridization of Mouse Transcripts
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Cells on glass coverslips (1.0 × 105 cells per well in a 24-well plate) were fixed using 4% PFA for 30 min and then permeabilized using 0.1% Triton X-100. The coverslips were then washed with PBS and soaked in a pre-hybridization buffer. The in situ hybridization (ISH) digoxigenin-labeled ENSMUST00000190863 or ENSMUST00000130679 probes (Boster, Pleasanton, CA, USA) were reconstituted in hybridization buffer (50% formamide, 10% dextran sulfate, 2× TSSC, 0.01% sheared salmon sperm DNA, and 0.02% SDS), and incubated at 37 °C overnight. Cells were washed twice with 2× saline sodium citrate (SSC) for 5 min at 37 °C, twice with 0.5× SSC for 15 min at 37 °C, and twice with 0.2× SSC for 5 min at 37 °C. After blocking with 5% normal goat serum for 1 h at 37 °C, the cells were incubated with anti-digoxigenin alkaline phosphatase conjugate (Roche Applied Science, Basel, Switzerland) and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium chloride buffer for 4 h. After DAPI staining, images were collected using a confocal microscope (Olympus).
3
Characterization of GhNSP gene expression
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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.
4
Linc-RA1 expression detection in glioma
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The expression of linc-RA1 in clinical glioma specimens was detected using ISH, performed as previously described49 (link). The sections were deparaffinized with xylene, rehydrated in serial dilutions of ethanol, and treated with 0.2 N HCL. After washing for 3 times, the sections were incubated in proteinase K (40 µg/mL, Promega) for 20 min and fixed with 4% paraformaldehyde for 10 min. The sections were reconstituted using hybridization solution and incubated at 56 °C overnight in a digoxigenin-labeled linc-RA1 probe (Exiqon, Vedbaek, Denmark). After washing, the sections were blocked with 5% normal goat serum for 1 h at room temperature followed by incubation in an anti-digoxigenin alkaline phosphatase conjugate (Roche, Stockholm, Sweden) overnight at 4 °C. Colorimetric signals were obtained by incubating the sections in 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro-blue tetrazolium chloride (NBT) buffer in the dark for 4 h at room temperature. Nuclear fast red was used as the counterstain.
5
In Situ Hybridization Protocol for H05 cDNA
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Briefly, a 150–200 bp fragment was amplified from the H05 cDNA. The PCR product was cloned into the pGEM‐T‐Easy vector (Promega, 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, Germany). Tissue sections were fixed in 50% FAA [10% formalin, 5% acetic acid, and 50% ethanol (v/v) in RNase‐free water]. After dehydration and embedding of the tissue in paraffin wax, the sample blocks were sectioned into 8‐µm slices using the microm HM 340E microtome (Thermo Scientific, USA) and were applied to RNase‐free glass slides (Solarbio, China). Hybridization was detected by using the antidigoxigenin‐alkaline phosphatase conjugate (Roche, dilution 1:1000), visualized by incubation with NBT/BCIP stock solution (Roche, dilution 1:50), and images were captured using a Zeiss Axioscope A1 microscope (Carl Zeiss, Germany). Primers are listed in Table S8 (Supporting Information).
6
In situ Hybridization of AhYSL3.1 in Peanut Roots
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In situ hybridization was performed as described previously [39 (link)]. Peanut roots grown hydroponically were fixed in formalin-acetic acid-alcohol and embedded in paraffin. The root tissues were sectioned into 10 μm slices and mounted on slides. The 3′UTR fragment of AhYSL3.1 was amplified using the primers: AhYSL3.1probe-F, 5′-TTTGCGATAGCAGCCAACTTGGTGAG-3′ and AhYSL3.1probe-R, 5′-AATTGTAGTTGCAAACTAGATACACTGATC-3′. After subcloning into the pCR®-Blunt II-TOPO® vector (Invitrogen, Carlsbad, CA, USA), the plasmid was linearized using BamHI. T7 RNA polymerase was used to generate sense and antisense probes, and the generated probes were labeled with digoxigenin-11-UTP (Roche, Mannheim, Germany). The slides were pretreated with proteinase K and subjected to acetylation and then hybridized for 16 h with the sense or antisense probe at 50 °C. After washing, the sections were incubated with anti-digoxigenin alkaline phosphatase conjugate (Roche) and stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche).
7
In Situ Hybridization for RIP140 Exon 1b
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An RNA probe for RIP140 exon 1b was transcribed from a cDNA template using a Megascript SP6 or T7 kit (Ambion), incorporating digoxigenin-11-UTP (Roche) (Nichol et al, 2006) . Sections were deparaffinised, rehydrated and then permeabilised by digestion with proteinase K. Sections were fixed in 4% paraformaldehyde, washed and air dried before hybridisation. The probe was diluted between 1:50-1:100 with hybridisation buffer (50% formamide, 5x SSC, 1x Denhardt's solution, 10% dextran sulphate, and 100 µg/ml denatured herring sperm DNA) and 10 µl of diluted probe was applied to each section and incubated in a humid chamber overnight at 55°C. RNase pre-treated sections or sections receiving hybridisation buffer only or sense probes, were used as negative controls. Following the hybridisation step, the slides were washed to remove excess and non-specifically bound riboprobe. For detection of the hybridised probe, slides were incubated with an anti-digoxigenin-alkaline phosphatase conjugate (Roche).Alkaline phosphatase-labelled hybrids were detected using a solution of 337 µg/ml nitro blue tetrazolium (NBT, Roche), 175 µg/ml 5-bromo-4-chloro-3indolyl phosphate (BCIP, Roche) and 1 mM levamisole. Slides were rinsed and mounted in aqueous mountant (Glycerol Gelatine, Sigma).
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