Whole-Mount In Situ Hybridization of Xenopus Oocytes

Cold Spring Harbor Protocols
Diana Bauermeister, Tomas Pieler

Abstract

Whole-mount in situ hybridization (WMISH) is a common approach that is used to visualize spatial and temporal gene expression in embryos. In this process, digoxygenin-labeled antisense RNA is hybridized to the complementary transcript of interest and RNA hybrids are immunohistochemically detected using an alkaline phosphatase-conjugated antibody against digoxigenin. During Xenopus laevis oogenesis, certain RNAs localize to the animal or vegetal pole laying the foundation for germ cell development and germ layer formation of the future embryo. Here we present a WMISH protocol for Xenopus laevis oocytes allowing for the efficient detection of localized RNAs in a large number of oocytes during different stages of oogenesis. The application of this approach might be combined with microinjection of tagged reporter RNAs and/or a gain- or loss-of-function background, allowing for the functional analysis of single protein factors involved in RNA localization.

References

May 22, 2009·The Journal of Biological Chemistry·Patrick K ArthurTomas Pieler
Jul 15, 2015·Developmental Biology·Diana BauermeisterTomas Pieler
Jan 13, 2018·Cold Spring Harbor Protocols·Tristan AgueroMary Lou King
Jan 13, 2018·Cold Spring Harbor Protocols·Karen NewmanMary Lou King

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Citations

Jan 26, 2020·Cold Spring Harbor Protocols·Kimberly L Mowry

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