Utilizing X-ray MicroCT to Develop a Robust Whole-Brain Staining Protocol

Mapping brain connectivity with volumetric electron microscopy relies on heavy metal staining. Existing heavy metal staining techniques can attain high membrane contrast, but only in relatively small volumes. Several approaches have been attempted to stain whole mouse brains, but they come with limitations such as incomplete staining, insufficient membrane contrast, or brain fragmentation. To systematically analyze the problems associated with current whole-brain staining methods, we leveraged X-ray microCT and time-lapse imaging. This approach helped us understand the reaction mechanisms, select appropriate reagents, efficiently determine optimal staining conditions, and importantly, identify the deleterious volume swelling and shrinkage that causes tissue damage. This effort led to a reliable and scalable whole-brain staining protocol that avoids tissue fragmentation, cracks, or weakly stained central areas.

Key Learnings:

• Systemic analysis of whole brain staining methods can be achieved using X-ray microCT and time-lapse imaging
• Using this approach, reaction mechanisms and optimal staining conditions can be understood and selected
• Reliable and scalable whole-brain staining protocols can be developed in this way