In situ hybridization experiments were performed as previously described (53 (link)). Antisense riboprobes were synthesized from linearized plasmids containing fragments of T. guttata coding sequences for candidate genes (primer sequences are provided in table S3). The Tbx5 probe was a gift from J. Gros (Pasteur Institute). Double in situ hybridizations were performed using riboprobes tagged with digoxigenin (DIG) or fluorescein RNA-labeling mix (Roche) and sequentially revealed using anti-DIG and anti-fluorescein alkaline phosphatase antibodies (Roche), followed by reaction on bromochloroindolyl phosphate–nitro blue tetrazolium (Promega) and Fast Red (Abcam) substrates.
Fast red
Manufactured by Abcam
Fast Red is a precipitating substrate that produces a red/pink colored reaction product when cleaved by alkaline phosphatase. It is commonly used in immunohistochemistry and in situ hybridization protocols.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using fast red
1
In Situ Hybridization of Avian Genes
2
Agouti and β-Catenin Expression Analysis
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In situ hybridization experiments were performed as described (24) using antisense riboprobes synthesized from vectors containing a 269-bp fragment of C. japonica, A. rufa, or S. reevesii's coding sequences for agouti, or an 881-bp fragment of C. japonica's coding sequence for b-catenin. For double in situ hybridizations, riboprobes were labeled with digoxigenin or fluorescein and sequentially revealed with anti-digoxigenin-AP or anti-fluorescein-AP antibodies (both 1:2000, Roche) and NBT/BCIP (Promega) or fast-red (Abcam) substrates.
Primers: agouti-F: TGCTCTGCTACAGTTTGCT-CAG; agouti-R: TGGTTTGCAGGTTTTGAA); b-catenin-F: AGCTGACTTGATGGAGTTGGA; b-catenin-R: TCGTGATGGCCAAGAATTTC).
Primers: agouti-F: TGCTCTGCTACAGTTTGCT-CAG; agouti-R: TGGTTTGCAGGTTTTGAA); b-catenin-F: AGCTGACTTGATGGAGTTGGA; b-catenin-R: TCGTGATGGCCAAGAATTTC).
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