Antisense riboprobes were generated using Roche DIG and FITC labeling mix. Portions of the coding regions of genes were PCR amplified using One Taq Hotstart 2X master mix in standard buffer DNA polymerase (NEB) and cloned into pCRII-TOPO TA vector (Thermofisher). Sequence verified clones were used to generate antisense riboprobes using appropriate enzymes. The zebrafish dnmt3bb.1 probe was generated as described previously13 (link). Whole mount in situ hybridization was carried out as described previously with a few modifications. To reduce non-specific hybridization and enhance signal to noise ratio we used 5% dextran sulfate (Sigma) in the hybridization buffer and pre-adsorbed anti-DIG and anti-FITC antibodies to whole cavefish powder. For histology, embryos and tissue samples were fixed using 4% para-formaldehyde overnight at 4°C and subsequently passed through ascending grades of alcohol followed by paraffin embedding. Sections were stained using hematoxylin and eosin (H&E).
Dig and fitc labeling mix
Manufactured by Roche
The DIG and FITC labeling mix is a laboratory reagent used for the detection and visualization of biomolecules such as DNA, RNA, and proteins. It contains two different labeling molecules, Digoxigenin (DIG) and Fluorescein isothiocyanate (FITC), which can be attached to target molecules for identification and analysis purposes. The DIG and FITC labeling mix is a versatile tool for researchers in various fields of biology and biochemistry.
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2 protocols using dig and fitc labeling mix
1
In situ Hybridization in Zebrafish
2
In situ Hybridization in Zebrafish
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Antisense riboprobes were generated using Roche DIG and FITC labeling mix. Portions of the coding regions of genes were PCR amplified using One Taq Hotstart 2X master mix in standard buffer DNA polymerase (NEB) and cloned into pCRII-TOPO TA vector (Thermofisher). Sequence verified clones were used to generate antisense riboprobes using appropriate enzymes. The zebrafish dnmt3bb.1 probe was generated as described previously13 (link). Whole mount in situ hybridization was carried out as described previously with a few modifications. To reduce non-specific hybridization and enhance signal to noise ratio we used 5% dextran sulfate (Sigma) in the hybridization buffer and pre-adsorbed anti-DIG and anti-FITC antibodies to whole cavefish powder. For histology, embryos and tissue samples were fixed using 4% para-formaldehyde overnight at 4°C and subsequently passed through ascending grades of alcohol followed by paraffin embedding. Sections were stained using hematoxylin and eosin (H&E).
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