Two-color FISH by combining POD and AP detection systems. 24-hpf embryos were simultaneously hybridized to differently-labeled pax6a and nkx6.1 probes and visualized by sequential recording of TSA-FAM (B,C,E,F) and Fast Red (A,C) or Fast Blue (D,F). Single channels (A,B,D,E) and overlays of two channels (C,F) are shown. Detection channels for Fast Blue, Fast Red and TSA-FAM were >650 nm, >560 nm, and 505 nm to 545 nm, respectively. Dotted lines indicate boundaries of basal part of prosomere 1. Abbreviations: b1, basal part of prosomere 1; b2, basal part of prosomere 2; m, midbrain; p1, prosomere 1; p2, prosomere 2, p3 prosomere 3; pr, pretectum; pt, prethalamus; t, telencephalon; th, thalamus. Images were recorded on a LSM510 confocal microscope and false-colored in ImageJ. Scale bar is 50 μm.