Abstract
In situ hybridization is a powerful method for detecting endogenous mRNA sequences in morphologically preserved samples. We provide in situ hybridization methods, which are specifically optimized for mouse embryonic samples as whole mounts and section tissues. Additionally, β-Galactosidase (β-gal) is a popular reporter for detecting the expression of endogenous or exogenous genes. We reveal that 6-chloro-3-indoxyl-β-d-galactopyranoside (S-gal) is a more sensitive substrate for β-gal activity than 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal). S-gal is advantageous where β-gal activity is limited including early stage mouse embryos. As a result of the increased sensitivity as well as the color compatibility of S-gal, we successfully combined β-gal staining using S-gal with in situ hybridization using DIG-labeled probes in both whole mounts and sections.
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References
Wilkinson DG (1992) In situ hybridization. A practical approach. Oxford University Press, New York, NY
Wilkinson DG, Nieto MA (1993) Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol 225:361–373
Varlet I, Collignon J, Robertson EJ (1997) Nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation. Development 124:1033–1044
Baker JC, Beddington RS, Harland RM (1999) Wnt signaling in Xenopus embryos inhibits bmp4 expression and activates neural development. Genes Dev 13:3149–3159
Kishigami S, Komatsu Y, Takeda H, Nomura-Kitabayashi A, Yamauchi Y, Abe K, Yamamura K, Mishina Y (2006) Optimized beta-galactosidase staining method for simultaneous detection of endogenous gene expression in early mouse embryos. Genesis 44:57–65
Acknowledgments
We thank Drs. Ken-ichi Yamamura for P0-Cre mice, and Philippe Soriano for Mox2-Cre and ROSA26 reporter mice; Drs. Andrew P. McMahon, Michael Rauchman, William Shawlot, Hiroshi Sasaki, Bernhard G. Herrmann, Terry P. Yamaguchi, Deborah L. Chapman, and Hiroshi Hamada for in situ probes; Dr. Gen Yamada for sharing with us the unpublished results; Ms. Trisha Castranio and Sudha Rajderkar for critical reading; Dr. Mark Lewandoski for generous editing of this manuscript. This work was supported by the National Institutes of Health (K99DE021054 to Y.K., R01DE020843 and ES071003-11 to Y.M.), a fellowship from the Japan Society for the Promotion of Science (Y.K.), and the conditional gift from RIKEN-Brain Science Institute (Y.M).
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Komatsu, Y., Kishigami, S., Mishina, Y. (2014). In Situ Hybridization Methods for Mouse Whole Mounts and Tissue Sections with and Without Additional β-Galactosidase Staining. In: Lewandoski, M. (eds) Mouse Molecular Embryology. Methods in Molecular Biology, vol 1092. Humana Press, Boston, MA. https://doi.org/10.1007/978-1-60327-292-6_1
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DOI: https://doi.org/10.1007/978-1-60327-292-6_1
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Publisher Name: Humana Press, Boston, MA
Print ISBN: 978-1-60327-290-2
Online ISBN: 978-1-60327-292-6
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