C-ECi: a CUBIC-ECi combined clearing method for three-dimensional follicular content analysis in the fish ovary†.

Deciphering mechanisms of oocyte development in the fish ovary still remain challenging, and a comprehensive overview of this process at the level of the organ is still needed. The recent development of optical tissue clearing methods has tremendously boosted the three-dimensional (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 nontoxic solvent-based ethyl cinnamate (ECi) method for efficient clearing and the Clear Unobstructed Brain Imaging Cocktails and Computational (CUBIC) method to enhance lipid removal and reduce nonspecific staining. The methyl green fluorescent dye was used to stain nuclei and delineate the follicular structures that include oocytes. Using this procedure (named CUBIC-ECi [C-ECi]), ovaries of both medaka and trout could be imaged in 3D and follicles analyzed. To our knowledge, this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-ECi method thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further developmental and morphological studies.