Whole Mount In Situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs- Chapter 14.9

PIZARD A; HARAMIS A; CARRASCO A; FRANCO P; LÓPEZ L; PAGANELLI A

Current Protocols in Molecular Biology

John Wiley & Sons, INC

Año: 2004; p. 1 - 5300

Whole-Mount In Situ Hybridization and UNIT 14.9UNIT 14.9 Detection of RNAs in Vertebrate Embryos and Isolated Organs The spatial and temporal distribution of transcripts (e.g., during embryogenesis) provides important clues to possible functions of the encoded gene products and/or possible interactions with other genes. The whole-mount in situ hybridization method, first introduced by Tautz and Pfeifle (1989) and applied to Drosophila embryos, has been extended to vertebrate embryos:Drosophila embryos, has been extended to vertebrate embryos: Xenopus (Harland, 1991; Franco et al., 1999), zebrafish (Schulte-Merker, 1993), and mouse and chicken (Wilkinson, 1993). Given the complex anatomy of embryos, the method simplifies detection and interpretation of gene expression patterns, especially for genes with graded or dynamic distribution. This method circumvents the tedious detection of RNA hybrids by emulsion autoradiography (UNIT 14.4) and subsequent reconstruction of their three-dimensional distribution. Whole-mount in situ hybridization using digoxigenin-labeled riboprobes is probably the most commonly used method to detect RNA transcripts in intact embryos or isolated organs (see Basic Protocol 1 and Alternate Protocol 1). The hybrids are subsequently detected with anti-digoxigenin antisera conjugated to alkaline phosphatase and immune complexes are visualized by an enzymatic reaction of alkaline phosphatase with a chromogenic substrate (see Basic Protocols 2 and 4 and Figs. 14.9.1, 14.9.2, and 14.9.3). The methods described in this unit are based on procedures by Riddle et al. (1993) and Wilkinson (1993). They have been used successfully for the detection of mRNAs in mouse and chicken embryos. The methods are also applicable to experimentally manipulated embryos—e.g., for detection of transcripts in mouse tissue grafted to chicken embryos (Haramis et al., 1995). Whole-mount in situ hybridization of Xenopus embryos requires different pretreatment, hybridization, and detection conditions because these embryos are yolk-rich and prone to high background (see Basic Protocols 3 and 4). Both hybridizations use digoxigeninlabeled antisense RNA probes (riboprobes; see Support Protocol 2), and hybridizations with mouse and chicken embryos use anti-digoxigenin antibody that will be preabsorbed with embryonic powder to reduce nonspecific binding of the detecting antibody to embryonic tissue (see Support Protocol 1).(Harland, 1991; Franco et al., 1999), zebrafish (Schulte-Merker, 1993), and mouse and chicken (Wilkinson, 1993). Given the complex anatomy of embryos, the method simplifies detection and interpretation of gene expression patterns, especially for genes with graded or dynamic distribution. This method circumvents the tedious detection of RNA hybrids by emulsion autoradiography (UNIT 14.4) and subsequent reconstruction of their three-dimensional distribution. Whole-mount in situ hybridization using digoxigenin-labeled riboprobes is probably the most commonly used method to detect RNA transcripts in intact embryos or isolated organs (see Basic Protocol 1 and Alternate Protocol 1). The hybrids are subsequently detected with anti-digoxigenin antisera conjugated to alkaline phosphatase and immune complexes are visualized by an enzymatic reaction of alkaline phosphatase with a chromogenic substrate (see Basic Protocols 2 and 4 and Figs. 14.9.1, 14.9.2, and 14.9.3). The methods described in this unit are based on procedures by Riddle et al. (1993) and Wilkinson (1993). They have been used successfully for the detection of mRNAs in mouse and chicken embryos. The methods are also applicable to experimentally manipulated embryos—e.g., for detection of transcripts in mouse tissue grafted to chicken embryos (Haramis et al., 1995). Whole-mount in situ hybridization of Xenopus embryos requires different pretreatment, hybridization, and detection conditions because these embryos are yolk-rich and prone to high background (see Basic Protocols 3 and 4). Both hybridizations use digoxigeninlabeled antisense RNA probes (riboprobes; see Support Protocol 2), and hybridizations with mouse and chicken embryos use anti-digoxigenin antibody that will be preabsorbed with embryonic powder to reduce nonspecific binding of the detecting antibody to embryonic tissue (see Support Protocol 1).UNIT 14.4) and subsequent reconstruction of their three-dimensional distribution. Whole-mount in situ hybridization using digoxigenin-labeled riboprobes is probably the most commonly used method to detect RNA transcripts in intact embryos or isolated organs (see Basic Protocol 1 and Alternate Protocol 1). The hybrids are subsequently detected with anti-digoxigenin antisera conjugated to alkaline phosphatase and immune complexes are visualized by an enzymatic reaction of alkaline phosphatase with a chromogenic substrate (see Basic Protocols 2 and 4 and Figs. 14.9.1, 14.9.2, and 14.9.3). The methods described in this unit are based on procedures by Riddle et al. (1993) and Wilkinson (1993). They have been used successfully for the detection of mRNAs in mouse and chicken embryos. The methods are also applicable to experimentally manipulated embryos—e.g., for detection of transcripts in mouse tissue grafted to chicken embryos (Haramis et al., 1995). Whole-mount in situ hybridization of Xenopus embryos requires different pretreatment, hybridization, and detection conditions because these embryos are yolk-rich and prone to high background (see Basic Protocols 3 and 4). Both hybridizations use digoxigeninlabeled antisense RNA probes (riboprobes; see Support Protocol 2), and hybridizations with mouse and chicken embryos use anti-digoxigenin antibody that will be preabsorbed with embryonic powder to reduce nonspecific binding of the detecting antibody to embryonic tissue (see Support Protocol 1).Xenopus embryos requires different pretreatment, hybridization, and detection conditions because these embryos are yolk-rich and prone to high background (see Basic Protocols 3 and 4). Both hybridizations use digoxigeninlabeled antisense RNA probes (riboprobes; see Support Protocol 2), and hybridizations with mouse and chicken embryos use anti-digoxigenin antibody that will be preabsorbed with embryonic powder to reduce nonspecific binding of the detecting antibody to embryonic tissue (see Support Protocol 1).