Tissue clearing techniques for three-dimensional reconstruction and imaging of entire organs and thick samples have become a popular and broadly used methodology, leading to the development of numerous protocols. Due to the complex cellular architecture of the brain and the wide spatial range of the connections that neurons may display, having the possibility to stain, image, and reconstruct neurons and/or neuronal nuclei in their entire extent can be crucial. However, this is hard to accomplish due to the natural opacity of the brain and the general thickness of the sample, posing a barrier to both imaging and antibody penetration. Nothobranchius furzeri has recently become a widely used model to study brain aging thanks to its short life span (3-7 mo), providing new opportunities to study the effects of aging on the brain and the role of aging in the development of neurodegenerative diseases. Here, we present a methodology to clarify and stain whole N. furzeri brains. This protocol is based on the ScaleA2 and ScaleS protocols developed and presented by Hama and colleagues, together with an in-house developed staining procedure for thick slices of tissues. ScaleS is a convenient and easy clearing technique based on sorbitol and urea that does not require particularly complex equipment, but due to the high urea concentration in some of the solutions, not all antigens are preserved. To overcome this issue, we developed a method that allows optimal staining of Nothobranchius furzeri brains before clarification.
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