Confocal Microscopy for Zebrafish Embryos

Fluorescent images were captured using an SP5 inverted confocal microscope (Leica) as previously described^{10 (link)}. For time-lapse imaging, embryos were embedded in agarose (1.0% in E3 medium) containing tricaine at a temperature of 28.5°C. z-stacks were taken every 196sec. Videos were created following processing with Volocity software (Improvision). acridine orange staining was performed as previously described^{32 (link)}. Briefly, embryos were dechorionated and incubated in 1X E3 medium containing 5μg/ml acridine orange (Sigma, A8097) for 30 min, followed by three washes in 1X E3. Embryos were then visualized by confocal microscopy. All images captured by confocal microscopy were displayed as maximum projections. Visible light imaging was performed on a BX-51 microscope using 100X oil objective lens and DP70 digital camera and software (Olympus) or a Leica MZ16 microscope and DFC295 digital camera and software (Leica).