Whole-mount in situ hybridization and double in situ hybridization were performed as previously described (Epstein et al., 1997 (link)). Embryos treated with DEAB (300 µM) or citral (70 µM) as well as untreated siblings, were fixed at stage 15 in 4% paraformaldehyde in MEM buffer (0.1 M MOPS, 0.5 M NaCl, 1 mM EGTA, 2 mM MgSO4), and processed for whole-mount in situ hybridization. Probes were prepared by in vitro transcription using Digoxigenin or Fluorescein labeling mix (Roche). Probes were transcribed as previously described: gsc (goosecoid) (Cho et al., 1991 (link)), sox3 (Penzel et al., 1997 (link)).
Fluorescein labeling mix
Manufactured by Roche
Fluorescein labeling mix is a reagent used to fluorescently label biomolecules, such as proteins, nucleic acids, or other compounds. It contains fluorescein, a commonly used fluorescent dye, and the necessary components for the labeling reaction. The core function of this product is to enable the covalent attachment of fluorescein to the target biomolecule, allowing for sensitive detection and visualization in various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein tyramide, by PerkinElmer (1 mentions)
Cy3 tyramide, by PerkinElmer (1 mentions)
Anti fluorescein hrp, by PerkinElmer (1 mentions)
Anti dig hrp, by PerkinElmer (1 mentions)
Sp5 2 scanning confocal microscope, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Axiovision imager a1, by Zeiss (1 mentions)
Bm purple, by Roche (1 mentions)
Magenta phosphate, by Merck Group (1 mentions)
4 protocols using fluorescein labeling mix
1
Embryonic Gene Expression Analysis
2
Dual-Fluorescence in situ Hybridization Protocol
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Whole-mount in situ hybridization (WISH) was done according to the protocol published previously (12 (link)). Double-fluorescence in situ hybridization (dFISH) was performed adapting the WISH protocol using the TSA signal amplification system (PerkinElmer). In short, additional to the DIG-labeled ISH probe, a second fluorescein-labeled probe was generated using fluorescein labeling mix (Roche). Probe concentration was reduced to 0.02 ng/µL for both probes. Anti–fluorescein-HRP (PerkinElmer) and anti–DIG-HRP (PerkinElmer) were diluted 1:500. Signal amplification was done with amplification diluent fluorescein-tyramide and amplification diluent Cy3-tyramide (PerkinElmer) for 4 min each. Samples were mounted in Vectashield (Vector Laboratories) and pictures were taken with a Leica SP5 II confocal scanning microscope. Stacks were acquired sequentially and z-projected.
3
Whole-Mount In Situ Hybridization Protocol
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RNA probes were produced with digoxigenin or fluorescein labeling mix (Roche). huc, her4, myoD, dlc, tbx24, her1 and mib plasmids were used as templates26 (link)44 (link)45 (link)46 (link)47 (link). The templates of dystrophin, foxc1a, fgf8a, papc, tcf15 and p53 were generated by RT-PCR with primers (listed in supplementary Table 6 ). The protocol of WISH followed the instruction of previous literatures48 (link)49 (link)50 (link). Embryos were mounted in 95% glycerol (Sigma, cat:15523)/PBST (UniRegion Bio-Tech, cat:UR-PBS001 and Tween20) pH 5.5 or flat-mounted in 95% glycerol/PBST pH 5.5 or 2:1 benzyl benzoate (Sigma, cat:B6630)/benzyl alcohol (Alfa Aesar, cat:041218). Images were captured by Zeiss Axiovision Imager A1 or Zeiss Discovery V8.
4
Whole-Mount In Situ Hybridization Protocols
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Whole-mount in situ hybridization and double in situ hybridization were performed as previously described (Epstein et al., 1997 (link)). Probes were prepared by in vitro transcription using Digoxigenin or Fluorescein labeling mix (Roche). Double staining was performed by either 5-Bromo-6-chloro-3-indolyl phosphate p-Toluidine salt (Magenta phosphate, Sigma) or BM purple (Roche) for the first probe and 5-Bromo-4-chloro-3-indolyl phosphate p-Toluidine salt (BCIP, Roche) for the second probe. Probes were transcribed as previously described: pax6 (Li et al., 1997 (link)), ncam1 (Krieg et al., 1989 (link)), muc2 (XCG-1) (Sive et al., 1989 (link)), chrd.1 (chordin) (Sasai et al., 1994 (link)), gsc (goosecoid) (Cho et al., 1991 (link)), cyp26a1 (Hollemann et al., 1998 (link)), aldh1a2 (raldh2) (Chen et al., 2001 (link)), aldh1a3 (raldh3) (Lupo et al., 2005 (link)), otx2 (Smith et al., 1993 (link)).
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