Detection of protein localization in combination with RNAscope. Embryos expressing either membrane-bound EGFP in their PGCs (A, B, C) or DsRed in their perinuclear germ-cell granules (D, E, F) were stained for vasa RNA at 20 hpf. vasa mRNA expression in the PGCs can be visualized (red in (A) and green in (D)) together with EGFP and DsRed ((B) and (E), respectively). The merged image of panels (A) and (B) is shown in (C) and that of (D) and (E) in panel (F). Anterior is to the left, dorsal up. A cross section of a 28-hpf wild-type zebrafish embryo showing the myoD transcript distribution detected using RNAscope (G), allowing the detection of E-cadherin by antibody staining (H). (I) Merged image of the two stainings. Scale bar corresponds to 20 μm. Dorsal is up. Single plane confocal images were captured using a 40× objective and subsequently processed as z-projections using the ImageJ software.