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).
Protocol detail
In situ Hybridization in Zebrafish
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Alcohol
Anti antibodies
Buffer
Clones
Dextran sulfate
Embryos
Enzymes
Eosin
Fitc
Formaldehyde
Genes
Hematoxylin
Hybridization
In situ hybridization
Powder
Taq dna polymerase
Tissue
Topo
Vector
Zebrafish
Corresponding Organization : Eunice Kennedy Shriver National Institute of Child Health and Human Development
Other organizations : University of Maryland, College Park
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Variable analysis
independent variables
- Use of Roche DIG and FITC labeling mix to generate antisense riboprobes
- PCR amplification of portions of coding regions using One Taq Hotstart 2X master mix in standard buffer DNA polymerase (NEB)
- Cloning of PCR amplified fragments into pCRII-TOPO TA vector (Thermofisher)
- Use of appropriate enzymes to generate antisense riboprobes from sequence verified clones
- Whole mount in situ hybridization patterns
- Histological staining (H&E) of embryos and tissue samples
- Use of previously described protocol for generating the zebrafish dnmt3bb.1 probe
- Use of 5% dextran sulfate in the hybridization buffer to reduce non-specific hybridization and enhance signal to noise ratio
- Pre-adsorption of anti-DIG and anti-FITC antibodies to whole cavefish powder
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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