To prepare the digoxigenin (DIG)-labeled RNA probes used for in situ hybridizations, cDNA fragments from the PLP (proteolipid protein), TUBB3 (class III-β-tubulin), and BLBP (brain lipid binding protein) genes were prepared as previously described.18 (link),35 (link) cDNA fragments for SOX2 (SRY-related HMG-box gene 2) (nucleotides 402–948 of GenBank: D50603.1) was obtained by PCR from E8 chick brain cDNA using specific primers based on GenBank sequences. Riboprobes incorporating DIG-labeled nucleotides were synthesized from linearized plasmid templates with SP6 or T7 polymerase (Roche). Slices of 40 μm were prepared for mRNA in situ hybridization as described previously.18 (link),35 (link),55 (link) After hybridization, DIG-labeled RNA duplexes were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche). Alkaline phosphatase activity was then detected using 5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) and nitro-blue tetrazolium (NBT) substrates (Roche) as described previously.18 (link),35 (link),55 (link)
Protocol detail
Preparation of DIG-labeled RNA Probes
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Alkaline phosphatase
Anti antibody
Based sequences
Brain
Brain lipid binding protein
Cdna
Digoxigenin
Genes
Hybridization
In situ hybridization
Mrna
Nitro blue tetrazolium
Nucleotides
Plasmid
Primers
Protein
Proteolipid
Rna probes
Sox2
Sry gene
Tubb3
Tubulin
Corresponding Organization :
Other organizations : Scripps Research Institute, University of Chicago
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Variable analysis
independent variables
- CDNA fragments from the PLP (proteolipid protein), TUBB3 (class III-β-tubulin), and BLBP (brain lipid binding protein) genes
- CDNA fragment for SOX2 (SRY-related HMG-box gene 2) (nucleotides 402–948 of GenBank: D50603.1)
- MRNA expression patterns of PLP, TUBB3, BLBP, and SOX2 genes
- Slices of 40 μm were prepared for mRNA in situ hybridization
- DIG-labeled RNA duplexes were detected with an alkaline phosphatase-conjugated anti-DIG antibody (Roche)
- Alkaline phosphatase activity was detected using 5-bromo-4-chloro-3′-indolyphosphate p-toluidine (BCIP) and nitro-blue tetrazolium (NBT) substrates (Roche)
- Not explicitly mentioned
- Not explicitly mentioned
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