Figure 6

Labeling of internal tissues by RNAscope probes. Detection of egfp mRNA employing RNAscope in 1-dpf Tg(elavl3:egfp) (A) (the white line marks the contours of the embryo) and 3-dpf Tg(fli1a:egfp) transgenic zebrafish embryos (B). Expression of egfp mRNA (red) and endogenous EGFP fluorescence (green) is shown in optical sections of 0 μm and 150 μm depth. The layer labeled as 0 μm was the first one where a signal was detected. The arrows mark internal vessels labeled in RNAscope. A graph showing the signal ratio of egfp RNA to EGFP protein fluorescence as measured in central parts of the embryo (an example of a selected area is presented with white outlines) at different depths of the tissue, indicates uniform labeling independent of section depth. Scale bars correspond to 100 μm. Single plane confocal images were captured using a 20× objective and 0.6× digital zoom in 3.5 to 4 μm intervals. Optical sections show merged images of three adjacent confocal planes. Sections were obtained from the lateral aspects of the embryo. Z-projections of all optical sections are shown on the right. Examining the RNA expression in 3-day-old larvae, egfp expression is detected in the lens, which does not exhibit EGFP signal, presumably reflecting non-specific staining of this structure. See also Additional files 8 and 9.