Control of bleed-through between Fast Red and TSA-FAM detection channels. 24-hpf embryos were hybridized to a dinitrophenol-labeled pax6a probe (A,B) or to a digoxigenin-labeled antisense RNA probe specific for nkx6.1 (C,D). Transcripts were detected using Fast Red (A,B) and TSA-FAM (C,D). Fluorescence signals were recorded in the Fast Red (Ch01: detection of wavelengths greater than 560 nm) and TSA-FAM (Ch02: detection of wavelengths from 505 nm to 545 nm) detection channels. A significant bleed-through of TSA-FAM nkx6.1 signal was detected in the Fast Red detection channel (C). Lateral views with anterior to the left are shown. Images were recorded on a LSM510 confocal microscope and false-colored in ImageJ. Scale bar is 50 μm.