In situ hybridization whole-mount

Judice TN, Nelson NC, Beisel CL, Delimont DC, Fritzsch B, et al. 2002. Cochlear whole mount in situ hybridization identification of longitudinal and radial gradients. Brain Res Prot 9 65-76. 

Large-Scale Whole Mount In Situ Hybridization of Mouse Embryos... 

Hume DA, Monkley SJ, Wainwright BJ. Detection of c-fms protooncogene in early mouse embryos by whole mount in situ hybridization indicates roles for macrophages in tissue remodelling. British Journal of Haematology 1995, 90, 939-942. 

Rosen B, Beddington R (1993) Whole-mount in situ hybridization in the mouse embryo gene expression in three dimensions. Trends Genet 9 162-167... 

Sample Preparation and Fixation Methods for Whole-Mount in Situ Hybridization... 

The general approach that we present for whole-mount in situ hybridization is shown schematically in Fig. 1 (see color plate). 

S rRNA metazoan phylogeny PCR with thermostable polymerase PCR with universal primers Whole mount in situ hybridization Engrailed antibody 4D9 Amphioxus Hox gene expression Amphioxus Hox gene cluster map 18S rDNA metazoan phylogeny Amphioxus ParaHox gene cluster... 

By 1990, however, it was possible to bring together all three of the advances discnssed above. First, technical advances (notably PCR and whole-mount in situ hybridization) meant... 

Tautz and Pfeifle Whole mount in situ hybridization... 

Fig. 3. Examples of the analysis of cultured explants. A Appearance of an explant in the stereomicroscope. The epithelium as well as the TGF(i-1-releasing bead have induced a translucent zone in dental mesenchyme. B Localization of cell proliferation with BrdU incorporation under the epithelium as well as around an FGF-4-releasing bead. C Whole-mount in-situ hybridization analysis of Msx-1 gene expression indicating induction by the epithelium in the mesenchyme. D Whole-mount immunohistochemical staining showing stimulation of tenascin expression in the mesenchyme around an FGF-4-releasing bead. E Section of an explant of dental mesenchyme and a TGF 3-1-releasing bead (the filter has been detached during processing). F Dark-field illumination of the explant in E showing the induction of tenascin-C transcripts by in-situ hybridization analysis e, dental epithelium m, dental mesenchyme b, bead.

Whole-Mount In-Situ Hybridization Combined with X-Gal Staining... 

Bell GW, Yatskievych TA, Antin PB (2004) GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos. Dev Dyn 229 677-87. Sang H (2004) Prospects for transgenesis in the chick. Mech Dev 121 1179-1186. Petitte JN, Liu G, Yang Z (2004) Avian pluripotent stem cells. Mech Dev 121 1159-1168. 

As the notochord is crucial for the development of the surrounding tissues but can be replaced by the floor plate of the neural tube (Fig. IB and IE), once this structure is established, the effect of notochord ablations varies depending on the type and timing of the operation. It is therefore advisable to analyze the embryos for the process of interest, but in addition for a known phenotype of the particular manipulation. This can be achieved, for instance, by double whole mount in-situ hybridization (see Fig. 1), employing molecular markers... 

The dye suggested here to stain the cell pellets remains visible after standard whole mount in-situ hybridization. 

HRP is not a fluorescent tracer (it generates a dense brown reaction product as outlined below), but has the advantage of producing permanent preparations and can be used in combination with whole-mount in situ hybridization techniques. 

Fig. 2. In-ovo electroporation. A pair of electrodes held by a manipulator (A) is inserted intom a window opened on the shell (B). The electrode is placed on the vitelline membrane overlying the embryo (C), and a 25-V 50-ms pulse is charged five times. The entire procedure is monitored under a dissection microscope. Plasmid solution is injected to the E2 (HH stage 10) chick neural tube (D) prior to the pulse charge.The dimensions of the electrode are shown in E. Most of the electrode is insulated (black in the figure) so that only the tip is exposed (white area). One hour after electroporation, some embryos were fixed, processed for paraffin sectioning, and observed with a Nomarski interference microscope (F). The right-hand side of the figure corresponds to the right of the embryo, where injected plasmid was transfected. The morphology of the cells and the structure of neural tube were almost normal. The blue deposit inside the neural tube is a complex of plasmid and the color substrates not removed by washing in dimethylformamide after whole-mount in-situ hybridization. Twenty-four hours after electroporation, the development of yolk sac plexus, vitelline veins, and vitelline arteries are retarded in the area contacted on the electrodes (arrows in G). Bar is 2mm (C) 50 pm (F) 4mm (G). Source (3).

The techniques for whole-mount in situ hybridization and antibody staining have been comprehensively reviewed elsewhere and, therefore, will not be discussed (Chapter 31 refs. 12,13). However, some general pointers and pitfalls for the selection of markers can be outlined. 

Jowett, T. and Lettice, L. (1994) Whole-mount in-situ hybridizations on zebrafish embryos using a mixture of digoxigenin-labeled and fluorescein-labeled probes. Trends Genet. 10,73,74. 

Wilkinson DG (1992) Whole mount in situ hybridization of vertebrate embryos. In Wilkinson DG (ed) In situ hybridization, a practical approach. IRL Press, Oxford University Press, Oxford, UK. 

Whole-mount in situ hybridization was first used to detect gene expression in Drosophila embryos (1). Various methods are now used to localize mRNAs in most species used for biological studies, and the methods have proven particularly useful when applied to vertebrate species. Here we provide a protocol commonly used for localizing transcripts in Xenopus embryos. The method is applicable to whole embryos and intact tissue explants. The methods are essentially as originally described by Hemmati-Brivanlou et al. (2) and then modified by HarlandfJ). 

Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98,81-85. Hemmati-Brivanlou, A., Frank, D., Bolce, M. B., Sive, H. L., and Harland, R. M. (1990) Localization of specific mRNAs in Xenopus embryos by whole mount in situ hybridization. Development 110,325-330. 

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