In situ hybridization was carried out on whole-mount embryos according to Moustakas (2008) and on paraffin-embedded sections according to Albrecht (‘97) . Digoxigenin- or 35S-labeled riboprobes were generated from linearized plasmids using T3 or T7 polymerase (Roche). For whole-mounts, mRNA expression was detected using alkaline phosphatase-coupled anti-digoxigenin antibody (Roche) and BM Purple (Roche). Turtle Bmp4 and Msx2 and chick Fgf8 probes were used with alligator embryos. Images of the radioactive in situ hybridization assays are the product of superimposing the pseudo-colored hybridization signal in Adobe Photoshop (Adobe, San Jose, CA) with a blue nuclear stain (Hoescht Stain, Sigma).
T3 or t7 polymerase
Manufactured by Roche
T3 or T7 polymerase is a laboratory enzyme used for in vitro transcription. It catalyzes the synthesis of RNA from a DNA template, which is a fundamental process in molecular biology and biotechnology.
Automatically generated - may contain errors
Lab products found in correlation
Bm purple, by Roche (1 mentions)
Hoescht stain, by Merck Group (1 mentions)
Alkaline phosphatase coupled anti digoxigenin antibody, by Roche (1 mentions)
Bcip nbt staining, by Vector Laboratories (1 mentions)
Fitc labeling mix, by Roche (1 mentions)
Dig rna labelling mix kit, by Roche (1 mentions)
Pdrive cloning vector, by Qiagen (1 mentions)
Nucleospin pcr clean up kit, by Macherey-Nagel (1 mentions)
4 protocols using t3 or t7 polymerase
1
In Situ Hybridization Assay for Embryonic Gene Expression
2
Whole-mount mRNA in situ Hybridization
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Digoxigenin-labeled antisense RNA probes were transcribed from linearized constructs using T3 or T7 polymerase (Roche). Whole-mount mRNA in situ hybridization was performed as described previously9 (link). The probes were detected using alkaline phosphatase (AP)-coupled anti-digoxigenin Fab fragment antibody (Roche) with BCIP/NBT staining (Vector Laboratories).
3
Xenopus Embryo Patterning Manipulation
4
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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