Anesthesia and Dissection Protocol for Ascidians

Animals were anesthetized using L-menthol based on a previously reported method^{19 (link),25 (link)}. In brief, 0.56% (weight per volume) L-menthol in ethanol was prepared and used as a stock solution. The stock solution was further diluted with 1% (volume per volume) artificial seawater before use. Animals were soaked in the diluted L-menthol solution for 10 min. Part of the tunic, body-wall muscle, and pharynx were excised under a fluorescence stereomicroscope (Leica M205 FA; Leica Microsystems, Wetzlar, Germany), and the whole animals were fixed in 4% paraformaldehyde in PBS at 4 °C overnight. The fixed specimens were soaked in 2 mg/ml glycine in PBS to quench the paraformaldehyde. The specimens were then washed three times with PBS and used for morphological analyses. Anatomic analyses were performed under the stereomicroscope (Leica M205 FA; Leica Microsystems) using precision tweezers (Outils Rubis SA, Stabio, Switzerland) and microscissors (Inami & Co., Ltd., Tokyo, Japan).