Analysis of 3D branching pattern: hematoxylin and eosin method.

Accurate analysis of the three-dimensional (3D) architecture of developing organs is critical to understanding how developmental defects can be linked with structural abnormalities. Here, we describe a 3D reconstruction technique of the developing kidney including the outer kidney capsule, ureteric epithelium, and developing nephrons. This 3D reconstructive process involves generating serial sections of the developing kidney, followed by histological staining. Each serial image is projected on the monitor and each tissue lineage or structure is traced. The kidney tracings are aligned and a 3D image is rendered. Each reconstructed tissue/lineage can then be subjected to quantitative analysis (e.g., surface area or volume). The reconstructed ureteric epithelium can be skeletonized to determine the branching architecture.