Szx10 fluorescent stereoscope

SEM used an adaptation of published methods [89 (link)]. In brief, embryos were fixed in 2.5% glutaraldehyde in PBS at 4 °C overnight, washed 3 × 10 min in 0.1 M PBS (pH 7.4) and transferred to 100% ethanol through a graded ethanol series. Specimens were then washed 5 × 10 min in 100% tertiary butyl alcohol and stored at 4 °C overnight. Samples were dried in a vacuum freeze dryer, placed on conductive tape, sprayed with platinum and observed under JSM-6390 scanning electron microscope. For GFP detection, amphioxus larvae were mounted in 1% methylcellulose in seawater and photographed under an SZX10 fluorescent stereoscope (Olympus, Japan) or an LSM 780 confocal microscope (Zeiss, Germany).

The posterior end of amphioxus larvae (with 3–4 gill slits) were amputated with a double-edged blade under a stereoscope. They were then raised in a petri dish (diameter = 6 cm) containing filtered sea water placed in a 30°C incubator and fed with Dicrateria zhangjiangensis twice a day. Sea water was changed once per day. After metamorphosis, they were moved to a 5 L plastic barrel and raised as previously described (Li et al., 2012 (link), 2014b (link)). Photographs were taken with an inverted microscope (Olympus, IX71) or an SZX10 fluorescent stereoscope (Olympus, Japan).