Trial of whole-organ and whole-body clearing in fish

Abstract

ScaleCUBIC-1, the newly-developed reagent for tissue clearing and decolorization, was trialed on the Malabar grouper (Epinephelus malabaricus), goldlined spinefoot (Siganus guttatus), and sapphire devil (Chrysiptera cyanea). After perfusion fixation with 4% paraformaldehyde (PFA) from the caudal vein of the Malabar grouper, tissues (brain, heart, spleen, liver, intestine, gill, eyes, and muscle) were dissected and post-fixed in 4% PFA at 4˚C for 24 hrs, before being immersed in ScaleCUBIC-1. Subsequently, the brain, spleen, intestine, gill, and muscle (but not skin or scale) were found to have been completely cleared, and the heart and liver partially so. Due to the thickness of the organ, deeper regions of the liver were not cleared. The failure of eye-clearing and decolorzation was partially due to pigmentation of the former. Similar results were obtained for tissues of the juvenile goldlined spinefoot and mature sapphire devil, following immersion in ScaleCUBIC-1 for 5 days. Whole body cleaning and decolorzation were also trialed on the goldlined spinefoot and sapphire devil. Following removal of the scales and skin, the immersion of specimens in ScaleCUBIC-1 for 14 days resulted in successful clearing and decolorzation of the muscles of both species. It was concluded that ScaleCUBIC-1 is suitable for clearing and decolorzation of fish tissues, including whole body, although modifications in the procedure may be required for different fish species.