Background

Protein expansion microscopy (proExM) is a powerful technique that crosslinks proteins to a swellable hydrogel to physically expand and optically clear biological samples. The resulting increased resolution (~70 nm) and physical separation of labeled proteins make it an attractive tool for studying the localization of subcellular organelles in densely packed tissues, such as the brain. However, the digestion and expansion process greatly reduce fluorescence signals making it necessary to optimize ExM conditions per sample for specific end goals.

New method

Here we compare the staining and digestion conditions of existing proExM workflows to identify the optimal protocol for visualizing subcellular organelles (mitochondria and the Golgi apparatus) within reporter-labeled neurons in fixed mouse brain tissue.

Results

We found that immunostaining before proExM and using a proteinase K based digestion for 8 h consistently resulted in robust fluorescence retention for immunolabeled subcellular organelles and genetically-encoded reporters.

Comparison with existing methods

With these methods, we more accurately quantified mitochondria size and number and better visualized Golgi ultrastructure in individual CA2 neurons in the mouse hippocampus.

Conclusions

This organelle optimized proExM protocol will be broadly useful for investigators interested in visualizing the spatial distribution of immunolabeled subcellular organelles in various reporter mouse lines, reducing effort, time and resources on the optimization process.

中文翻译：

---

蛋白质保留扩展显微镜用于观察固定脑组织中的亚细胞细胞器

背景

蛋白质膨胀显微镜 (proExM) 是一种强大的技术，可将蛋白质与可膨胀水凝胶交联，从而物理膨胀并光学透明的生物样品。由此产生的分辨率提高（~70 nm）和标记蛋白质的物理分离使其成为研究大脑等密集组织中亚细胞细胞器定位的有吸引力的工具。然而，消化和扩增过程大大减少了荧光信号，因此有必要优化每个样品的 ExM 条件以实现特定的最终目标。

新方法

在这里，我们比较了现有 proExM 工作流程的染色和消化条件，以确定在固定小鼠脑组织中报告标记的神经元内可视化亚细胞细胞器（线粒体和高尔基体）的最佳方案。

结果

我们发现，在 proExM 之前进行免疫染色并使用基于蛋白酶 K 的消化 8 小时，一致地导致免疫标记的亚细胞细胞器和基因编码报告基因的强荧光保留。

与现有方法的比较

通过这些方法，我们更准确地量化了小鼠海马单个 CA2 神经元中线粒体的大小和数量，并更好地可视化了高尔基体超微结构。

结论

这种细胞器优化的 proExM 协议对于有兴趣可视化各种报告小鼠系中免疫标记亚细胞细胞器的空间分布的研究人员将广泛有用，从而减少优化过程中的工作量、时间和资源。