Background

Although tissue clearing and subsequent whole-brain imaging is now possible, standard protocols need to be adjusted to the innate properties of each specific tissue for optimal results. This work modifies exiting protocols to clear fragile brain samples and documents a downstream pipeline for image processing and data analysis.

New Method

We developed a clearing protocol, CUBIC-f, which we optimized for fragile samples, such as the salamander brain. We modified hydrophilic and aqueous’ tissue-clearing methods based on Advanced CUBIC by incorporating Omnipaque 350 for refractive index matching.

Results

By combining CUBIC-f, light sheet microscopy and bioinformatic pipelines, we quantified neuronal cell density, traced genetically marked fluorescent cells over long distance, and performed high resolution characterization of neural progenitor cells in the salamander brain. We also found that CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains.

Comparison with exiting methods

CUBIC-f shortens clearing and staining times, and requires less reagent use than Advanced CUBIC and Advanced CLARITY.

Conclusion

CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains, larval and adult salamander brains which display considerable deformation using traditional CUBIC and CLARITY protocols.

中文翻译：

---

CUBIC-f：一种优化的清除方法，可用于追踪brain和评估brain脑中的神经突密度

背景

尽管现在可以进行组织清除和随后的全脑成像，但需要针对每个特定组织的先天特性调整标准方案，以获得最佳结果。这项工作修改了现有协议以清除易碎的大脑样本，并记录了用于图像处理和数据分析的下游管道。

新方法

我们开发了一种清除协议CUBIC-f，该协议已针对脆弱的样本（如the脑）进行了优化。我们通过结合Omnipaque 350进行折射率匹配，改进了基于先进CUBIC的亲水性和水性组织清除方法。

结果

通过结合CUBIC-f，光片显微镜和生物信息学流水线，我们量化了神经元细胞密度，在远距离上追踪了遗传标记的荧光细胞，并对the脑中的神经祖细胞进行了高分辨率表征。我们还发现CUBIC-f适用于保存胚胎小鼠大脑中的组织完整性。

与现有方法的比较

与Advanced CUBIC和Advanced CLARITY相比，CUBIC-f缩短了清洁和染色时间，并减少了试剂用量。

结论

CUBIC-f适用于保存胚胎小鼠的大脑，幼虫和成年sal脑的组织完整性，而使用传统的CUBIC和CLARITY协议它们会表现出很大的变形。