C-Eci: A Cubic-Eci Combined Clearing Method For 3D Follicular Content Analysis In The Fish Ovary

Deciphering mechanisms of oocyte development in female fishes still remains challenging and a comprehensive overview of this process at the level of the organ is still needed. The recent development optical tissue clearing methods have tremendously boosted the 3D imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the non-toxic solvent-based Eci method for efficient clearing and the CUBIC method to enhance lipid removal and reduce non-specific staining. The methyl green fluorescent dye was used to stain nuclei and delineate follicles. Using this procedure (named C-Eci), ovaries of both medaka and trout could be imaged in 3D and all follicles analyzed. To our knowledge this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-Eci methods thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further morphological studies. Summary A modified ethyl-cinnamate-based clearing method allows solving fish ovary-specific challenges for 3D imaging, including high lipid-contents, and analyzing the ovarian follicular content in medaka and trout.