Abstract
We report a dual fluorescent in situ hybridization (FISH) method for direct comparison of cellular distributions of different gene transcripts in the embryonic zebrafish brain and other tissues. After simultaneous hybridization of two differently labeled antisense RNA probes, the different hapten labels are visualized by peroxidase (POD)-mediated deposition of fluorochrome-labeled tyramides and alkaline phosphatase-based Fast Blue or Fast Red chromogenic staining, respectively. Since chromogenic Fast Blue and Fast Red precipitates display red-fluorescent emission, multiplexed visualization of different transcripts is possible by combination with carboxyfluorescein-labeled tyramides, which show emission in the green spectrum. The application of differential reporter enzymes provides advantages over procedures using sequential POD detection. The POD-coupled and AP-coupled antibodies can be mixed together in a single incubation step reducing the required time, spent otherwise for extensive washings and multiple incubations. In addition, removal or inactivation of antibody-POD conjugates as required in sequential POD detection procedures can be omitted. Therefore, potential false-positive detection of co-localization by insufficient inactivation is prevented.
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References
Hauptmann G, Gerster T (1996) Multicolour whole-mount in situ hybridization to Drosophila embryos. Dev Genes Evol 206(4):292–295. doi:10.1007/s004270050055
Hauptmann G, Gerster T (2000) Multicolor whole-mount in situ hybridization. Methods Mol Biol 137:139–148. doi:10.1385/1-59259-066-7:139
Hauptmann G, Gerster T (1994) Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet 10(8):266 doi:10.1016/0168-9525(90)90008-T
Spaniol P, Bornmann C, Hauptmann G, Gerster T (1996) Class III POU genes of zebrafish are predominantly expressed in the central nervous system. Nucleic Acids Res 24(24):4874–4881. doi:10.1093/nar/24.24.4874
Kitambi SS, Hauptmann G (2007) The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain. Gene Expr Patterns 7(4):521–528. doi:10.1016/j.modgep.2006.10.006
Hauptmann G, Gerster T (2000) Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech Dev 91(1–2):105–118, doi: 10.1016/S0925-4773(99)00277-4
Hauptmann G, Söll I, Gerster T (2002) The early embryonic zebrafish forebrain is subdivided into molecularly distinct transverse and longitudinal domains. Brain Res Bull 57(3–4):371–375, doi: 10.1016/S0361-9230(01)00691-8
Hauptmann G, Belting HG, Wolke U, Lunde K, Söll I, Abdelilah-Seyfried S, Prince V, Driever W (2002) spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation. Development 129(7):1645–1655
Hauptmann G, Gerster T (1995) Pou-2—a zebrafish gene active during cleavage stages and in the early hindbrain. Mech Dev 51(1):127–138, doi: 10.1016/0925-4773(95)00360-D
Bräutigam L, Hillmer JM, Söll I, Hauptmann G (2010) Localized expression of urocortin genes in the developing zebrafish brain. J Comp Neurol 518(15):2978–2995. doi:10.1002/cne.22375
Chandrasekar G, Lauter G, Hauptmann G (2007) Distribution of corticotropin-releasing hormone in the developing zebrafish brain. J Comp Neurol 505(4):337–351. doi:10.1002/cne.21496
Holzschuh J, Hauptmann G, Driever W (2003) Genetic analysis of the roles of Hh, FGF8, and nodal signaling during catecholaminergic system development in the zebrafish brain. J Neurosci 23(13):5507–5519
Hauptmann G (2001) One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos. Methods 23(4):359–372. doi:10.1006/meth.2000.1148
Speel EJ, Ramaekers FC, Hopman AH (1995) Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry, “who is still afraid of red, green and blue?”. Histochem J 27(11):833–858
Speel EJ (1999) Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors. Histochem Cell Biol 112(2):89–113
Lauter G, Söll I, Hauptmann G (2013) Molecular characterization of prosomeric and intraprosomeric subdivisions of the embryonic zebrafish diencephalon. J Comp Neurol 521(5):1093–1118. doi:10.1002/cne.23221
Denkers N, Garcia-Villalba P, Rodesch CK, Nielson KR, Mauch TJ (2004) FISHing for chick genes: triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos. Dev Dyn 229(3):651–657. doi:10.1002/dvdy.20005
Lauter G, Söll I, Hauptmann G (2014) Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification. Methods Mol Biol 1082:175–185. doi:10.1007/978-1-62703-655-9_12
Lecuyer E, Parthasarathy N, Krause HM (2008) Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. Methods Mol Biol 420:289–302. doi:10.1007/978-1-59745-583-1_18
Speel EJ, Hopman AH, Komminoth P (2006) Tyramide signal amplification for DNA and mRNA in situ hybridization. Methods Mol Biol 326:33–60
Lauter G, Söll I, Hauptmann G (2011) Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. BMC Dev Biol 11:43. doi:10.1186/1471-213X-11-43
Cowles MW, Brown DD, Nisperos SV, Stanley BN, Pearson BJ, Zayas RM (2013) Genome-wide analysis of the bHLH gene family in planarians identifies factors required for adult neurogenesis and neuronal regeneration. Development 140(23):4691–4702
Lauter G, Söll I, Hauptmann G (2011) Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Neural Dev 6(1):10. doi:10.1186/1749-8104-6-10
Acknowledgments
Imaging was performed at the Live Cell Imaging Unit, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, and supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Center for Biosciences.
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Hauptmann, G., Lauter, G., Söll, I. (2015). Application of Alkaline Phosphatase-Mediated Azo Dye Staining for Dual Fluorescent In Situ Hybridization in Zebrafish. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_20
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DOI: https://doi.org/10.1007/978-1-4939-2303-8_20
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