Comparison of conventional ISH methods with RNAscope. Using the myoD antisense probe to label muscle tissue, whole-mount 24-hpf zebrafish embryos were subjected to chromogenic ISH (A, B), FISH (C–F) (arrows in (C) and (E) indicate non-specific background in the yolk extension) and RNAscope-based mRNA detection (G, H). FISH was either performed using the standard (C, D) or optimized conditions (E, F). For panels (A)–(H), anterior is to the left and dorsal up. Images were captured using the indicated objectives. (I)
myoD mRNA is detected as distinct spots (shown in red) in the nuclei (arrows) and in the cytoplasm of the cells. Two different optical sections of the same sample from Additional file 7 are displayed. (J) The localization of vasa RNA in the cleavage furrows of a four-cell stage embryo as visualized using the RNAscope technology (red), with the nuclei stained by DAPI (blue). Scale bars for (A)–(H) and (J): 50 μm and for (I): 5 μm. Confocal images of whole-mount zebrafish embryos were captured in 3-μm intervals and processed either as z-projections (for (A)–(H), and (J)) or as different focal planes (for (I)) using the ImageJ software. Images (A)–(H) were captured and subsequently processed using conditions for achieving the best image for each method.