Application of Hydrogen Peroxide-Melanin Bleaching and Fluorescent Nuclear Staining for Whole-Body Clearing and Imaging in Fish

Abstract

Recently, our lab reported that CUBIC, a tissue-clearing technique, could help reveal the initial route of infection, its spread, and the localization of pathogens in infected goldfish Carassius auratus. ​However, this technique, in its original form, failed to clear melanin pigmented parts of the fish. ​We show here that bleaching with H₂O₂ clears melanin pigments of Black Moor goldfish without causing severe histological damage. ​Furthermore, nuclear staining with PI helps visualize the internal structures of cleared fish in a dark field. ​Since bleaching does not significantly quench the fluorescence of GFP, it may be applied to the in vivo imaging of GFP.