All animals were anesthetized in cool seawater (15°C) mixed with 2% MgCl2 (Chem-Supply, Australia) and sacrificed by an overdose of MgCl2. The specimens were then soaked into 10% neutral formalin or 4% paraformaldehyde (PFA) (EM grade, Electron Microscopy Sciences, Hatfield, USA) for at least 48 h and then transferred to 0.1% PFA-PBS fixative for storage at 4°C until further dissection for gross anatomy examination and histology.
Three adults of S. plangon (ML = 71–107 mm) and five juveniles of S. lessoniana (ML = 40–113 mm) for MR imaging were fixed using the transcardial perfusion protocol developed by Chung, et al.63 (link) In brief, the animals were anesthetized in cool seawater (15°C) mixed with 2% MgCl2 and sacrificed by an overdose of MgCl2prior to fixation. The transcardial perfusion protocol is using 4% PFA mixed with 0.1 M PBS with the rate of perfusion set to 2.5 mL per minute. The perfusion proceeded until 0.2 mL fixative per gram of specimen was used. Subsequently the muscle, skin and connective tissues around the brain were removed and the specimen was soaked in 4% PFA-PBS fixative for overnight to reduce morphological deformation of the brain.
Three adults of S. plangon (ML = 71–107 mm) and five juveniles of S. lessoniana (ML = 40–113 mm) for MR imaging were fixed using the transcardial perfusion protocol developed by Chung, et al.63 (link) In brief, the animals were anesthetized in cool seawater (15°C) mixed with 2% MgCl2 and sacrificed by an overdose of MgCl2prior to fixation. The transcardial perfusion protocol is using 4% PFA mixed with 0.1 M PBS with the rate of perfusion set to 2.5 mL per minute. The perfusion proceeded until 0.2 mL fixative per gram of specimen was used. Subsequently the muscle, skin and connective tissues around the brain were removed and the specimen was soaked in 4% PFA-PBS fixative for overnight to reduce morphological deformation of the brain.