Plastic embedding for precise imaging of large-scale biological tissues labeled with multiple fluorescent dyes and proteins.

Resin embedding of multi-color labeled whole organs is the primary step to preserve structural information for visualization of fine structures in three dimensions. It is essential to study the morphological characteristics, spatial and positional relationships of the millions of neurons, and the intricate network of blood vessels with fluorescent labels in the brain. However, the current resin embedding method is inadequate because of incompatibilities with fluorescent dyes, making it difficult to reconstruct a variety of structures for the interpretation of their complex spatial relationships. We modified the resin embedding method for large biological tissues labeled with multiple fluorescent dyes and proteins through different labeling strategies. With TrueBlack as the background fluorescence inhibitor in the glycol methacrylate (GMA) embedding, we referred to the method as GMA-T (Glycol methacrylate with TB). In the GMA-T embedded mouse brains, structures labeled with fluorescent proteins and dyes were visualized in millimeter-scale networks with sub-cellular resolution, allowing quantitative analysis of different anatomical structures in the same brain, including neurons and blood vessels. In combination with high-resolution whole-brain imaging, it is possible to obtain a variety of fluorescence labeled structures in just a few days. We quantified the distribution and morphology of the tdTomato-labeled vasoactive intestinal polypeptide (VIP) neurons and the BSA-FITC labeled blood vessels in the same brain. These results demonstrated that VIP neurons and blood vessels have their own unique distribution patterns and morphological characteristics among cortical regions and different layers in cerebral cortex, and there was no significant correlation between VIP neurons and vessels. This approach provides a novel approach to study the interaction among different anatomical structures within large-volume biological samples labeled with multiple fluorescent dyes and proteins, which helps elucidating the complex anatomical characteristics of biological organs.