Zebrafish embryos were fixed in 4% PFA at 4 °C O/N. Digoxigenin (DIG)-labeled antisense probes were synthesized with DIG RNA labelling MIX kit (Roche) using a DNA template amplified from cDNA using specific primers: snap29 T3 5′-taatacgactcactatagggagaATGTCTGCCTACCCCAAATC-3′, snap29 T7 5′-attaaccctcactaaagggagaACATCTCATCCAGGTTTCT-3′. After hybridization, detection was performed with anti-DIG antibody coupled to alkaline phosphatase (Roche) and specimen were imaged with Olympus SZX12 stereomicroscope.
Dig rna labelling mix kit
Manufactured by Roche
Sourced in France
The DIG RNA labelling MIX kit is a product designed for the non-radioactive labelling of RNA. It contains the necessary components to incorporate digoxigenin-UTP into RNA transcripts during in vitro transcription. This allows for the sensitive detection of the labelled RNA through immunological methods.
Automatically generated - may contain errors
2 protocols using dig rna labelling mix kit
1
Whole-Mount In Situ Hybridization in Zebrafish
2
In Situ Hybridization of Soff-IR25
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The amplified fragment of Soff-IR25 was purified with a NucleoSpin PCR Clean-Up kit (Machery-Nagel, Germany), cloned into pDrive Cloning Vector (Qiagen,Valencia, CA, USA) and sequenced by Eurofin Biotech (Germany). RNA probes were then generated by in vitro transcription using digoxigenin-11-UTP (Dig RNA labelling mix kit, Roche, Meylan, France). Antisense and sense probes were obtained with T3 or T7 polymerase (Roche). RNA probes were purified by cold precipitation with lithium chloride and anhydrous alcohol.
Sense RNA probes were used as negative controls. In situ hybridizations were performed on MABT and left at 4°C in an AP buffer (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 0.1% Tween-20) overnight. Finally, AP buffer, with 50 mM MgCl 2 , was added to the embryos twice for 30 min each before development of staining with the addition of 100 μg/mL 5bromo-4-chloro-3-indolyl phosphate (BCIP) and 80 μg/mL nitroblue tetrazolium chloride (NBT) until the desired contrast was reached. After several PBS rinses, embryos were postfixed with 3.7% formaldehyde for 24h, and finally rinsed with PBS. The total number of embryos used for ISH, including controls, was 6.
For histological visualization of staining, hybridized embryos were impregnated in 0.12M phosphate buffer pH 7.2 with 15% saccharose at 4°C for twice 24 h. Then, they were
Sense RNA probes were used as negative controls. In situ hybridizations were performed on MABT and left at 4°C in an AP buffer (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 0.1% Tween-20) overnight. Finally, AP buffer, with 50 mM MgCl 2 , was added to the embryos twice for 30 min each before development of staining with the addition of 100 μg/mL 5bromo-4-chloro-3-indolyl phosphate (BCIP) and 80 μg/mL nitroblue tetrazolium chloride (NBT) until the desired contrast was reached. After several PBS rinses, embryos were postfixed with 3.7% formaldehyde for 24h, and finally rinsed with PBS. The total number of embryos used for ISH, including controls, was 6.
For histological visualization of staining, hybridized embryos were impregnated in 0.12M phosphate buffer pH 7.2 with 15% saccharose at 4°C for twice 24 h. Then, they were
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