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Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos

Nature Protocols volume 4, pages 975–983 (2009)Cite this article

Abstract

In situ hybridization (ISH) is widely used to study the spatial distribution of gene expression in developing embryos. It is the method of choice to analyze the normal pattern of expression of a gene and also to characterize how the expression of a gene, or a group of genes, is altered in response to experimental or genetic manipulations. The standard protocols for this technique use a chromogenic reaction that produces a purple or red precipitate in cells expressing the target gene. This technique has significant disadvantages when compared with fluorescent techniques, as it cannot detect regions of overlap and external staining masks internal staining. We present a protocol for three-channel fluorescent ISH (FISH) optimized for wholemount analysis of large vertebrate embryos. Multichannel FISH in combination with immunofluorescence or chromogenic ISH offers a suite of approaches that allow accurate mapping of overlapping gene expression patterns in two- and three-dimensions. The time required for the protocol varies depending on the number of channels sampled and ranges from 3 to 5 d plus an additional 2 d to completely wash embryos and prepare for documentation.

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Figure 1: Two-channel FISH combined with immunofluorescence.
Figure 2: FCIS staining of slc4a4 (BM purple) and atp1a1 (FITC).
Figure 3: Optical transverse section of wholemount FISH staining for pax8 in the developing kidney.
Figure 4: Confocal analysis of wholemount FISH and double FISH.
Figure 5
Figure 6: Expected results by wholemount FISH.

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Acknowledgements

These protocols are adaptations of those originally developed by Richard Harland and Lance Davidson. The Photoshop image manipulation pipeline was developed by Victor Gerth, who also performed the data processing for the 3D models. This work was supported by grants from the AHFMR to P.D.V.

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Authors and Affiliations

  1. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

    Peter D Vize, Kyle E McCoy & Xiaolan Zhou

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  1. Peter D Vize
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Corresponding author

Correspondence to Peter D Vize.

Supplementary information

Supplementary Movie 1: Two channel FISH sampled in 3D.

Two channel FISH illustrating expression of nephrin in the pronephric glomus and atp1a1 in the pronephric nephron. Sampling by confocal microscopy. (MOV 1334 kb)

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Vize, P., McCoy, K. & Zhou, X. Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. Nat Protoc 4, 975–983 (2009). https://doi.org/10.1038/nprot.2009.69

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