Laboratory of Biology (BIO@SNS), Scuola Normale Superiore, 56126, Pisa, Italy.
Biology and Evolution of Marine Organisms Dep. (BEOM), Stazione Zoologica Anton Dohrn, 80121, Napoli, Italy.
Cold Spring Harb Protoc. 2023 Sep 1;2023(9):698-704. doi: 10.1101/pdb.prot107793.
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. 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 brains. This protocol is based on the ScaeA2 and ScaeS protocols developed and presented by Hama and colleagues, together with an in-house developed staining procedure for thick slices of tissues. ScaeS 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 brains before clarification.
组织透明化技术已成为一种广泛应用的方法，可用于三维重建和整个器官及厚样本的成像，由此产生了许多不同的方案。由于大脑的细胞结构复杂，神经元之间的连接范围广泛，因此有可能对神经元和/或神经元核进行全范围染色、成像和重建至关重要。然而，由于大脑的天然不透明度和样本的总体厚度，这很难实现，这对成像和抗体渗透都构成了障碍。秀丽隐杆线虫最近成为研究大脑衰老的广泛使用模型，因为它的寿命短（3-7 个月），为研究衰老对大脑的影响以及衰老在神经退行性疾病发展中的作用提供了新的机会。在这里，我们提出了一种对整个秀丽隐杆线虫大脑进行透明化和染色的方法。该方案基于 Hama 及其同事开发和提出的 ScaeA2 和 ScaeS 方案，以及我们自己开发的用于厚组织切片的染色程序。ScaeS 是一种基于山梨醇和尿素的方便且简单的透明化技术，不需要特别复杂的设备，但由于一些溶液中的尿素浓度较高，并非所有抗原都能保留下来。为了解决这个问题，我们开发了一种在透明化之前允许秀丽隐杆线虫大脑进行最佳染色的方法。
