Whole Mount in situ Hybridization Reveals mRNA Expression

To study mRNA expression, whole mount in situ hybridization was performed using sense and antisense riboprobes prepared with a digoxigenin RNA labelling kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. At least three mouse E9.5 embryos (25–30 somites) were analyzed per probe, as described previously (Ybot-Gonzalez et al., 2005 (link)). Selected embryos, labelled for Daam1 (Ybot-Gonzalez et al., 2007 (link)), glypican4 (Ybot-Gonzalez et al., 2005 (link)) or Wnt5a (Gavin et al., 1990 (link)) were embedded in a gelatin-sucrose-albumin and glutaraldehyde solution, and vibratome sections (50 μm) of the embryos were photographed by using an Axiophot (Zeiss, Jena, Germany) photomicroscope. To compare Daam1 expression between control and treated embryos, the in situ hybridization procedure was performed in only one tube, adequately marking the embryos for their later identification. Sense-strand control riboprobes gave no specific signal.
Embryos at E11.5 and E12.5 were fixed in 4% PFA, and they were immediately whole mount stained using the X-Gal method (Carroll et al., 2003 (link)). Eosin counterstained paraffin sections (7 μm) were examined.