Application of Tissue Clearing Techniques to 3D Study of Infectious Disease Pathology in Fish

Conventional fish pathological diagnoses are limited in ability to detect pathogens in infected fish due to limitations of partial or 2 dimensional images. Here, we tested three tissue clearing techniques (ClearT, SeeDB and CUBIC), which make the fish body transparent, allowing visualization of pathogens. CUBIC was most effective in clearing the fish body, and was applicable to immunohistochemistry and GFP (green fluorescent protein) imaging. Furthermore, DAPI-labeled Aeromonas hydrophila was visible under a fluorescence microscope. Thus, CUBIC is the most suitable tissue clearing technique allowing 3 dimensional analysis of infectious diseases in fish.