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E17.5 or E18.5 embryos were sacrificed by making a cut in the neck to sever the spinal cord, and tail tips were taken for genotyping. After incubating the embryos for 2 min each in an increasing and then decreasing methanol series and washing in 1× PBS, they were stained in a 0.1% toluidine blue solution in water for 1–2 min on ice. A specific dye pattern showing possible barrier defects appeared after de-staining the embryos in 1× PBS on ice7,72.
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