Understanding 3-Dimensional World from 2-Dimensional Immunofluorescent Adjacent Sections

In many fields of biological sciences including embryology and cancer research, understanding of 3-dimensional structures is crucial to uncovering normal and pathological phenomena. While the most optimal method would be to directly observe the complete object without any destruction, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues stained each with chromogenbased immunohistologicalmethods and painstakingly reconstructed the 3-dimensional volume. The limiting factor with immunohistological staining is the difficulty in detecting multiple antigens with different chromogens on the same tissue. At our Molecular Cytology Core Facility at Memorial Sloan-Kettering Cancer Center, we successfully and routinely perform immunofluorescent staining using automated staining machines and have combined IF staining and 3D reconstruction of serial sections.This method allows simultaneous detection of up to four different antigens on the same sections in a highly reproducible and specific manner. The resulting stack can be a stunning visualization of 3D structure and be quantitatively analyzed.