XB-ART-39821Nat Protoc 2009 Jan 01;46:975-83. doi: 10.1038/nprot.2009.69.
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Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos.
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.
PubMed ID: 19498377
Article link: Nat Protoc
Species referenced: Xenopus
Genes referenced: atp1a1 slc12a1 tbx2
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References [+] :
Acloque, In situ hybridization analysis of chick embryos in whole-mount and tissue sections. 2008, Pubmed