Detection of rare transcripts with RNAscope. Using chromogenic ISH, 24-hpf embryos were stained for cxcr4b mRNA for short (2 hours at RT) (A, B) or long (1 day at RT) (C) staining durations. After a short staining reaction, the expression of cxcr4b is not detected in the PGCs (B, arrow). Prolonged staining of the embryos resulted in weak staining in the PGCs (C, arrow). Using the RNAscope protocol with 24-hpf embryos, the weak cxcr4b expression in the gonad region (D, arrow) was detected in the PGCs that also expressed the nanos transcript (E, arrow). (F) Weak expression of cxcl12a in the region of the PGCs is observed as well as strong expression along the lateral line of the embryo. An overlay image of panels (D)–(G) is shown in (H). Scale bars in (B) and (C) correspond to 200 μm and that in (H) is for (D)–(H) and signifies 50 μm. For all panels, anterior is left and dorsal up. Confocal images were captured at 3-μm intervals and subsequently processed as z-projections using the ImageJ software.