NOMIS researcher Ali Ertürk and colleagues have developed a new chemical method, wildDISCO, that uses conventional antibodies and fluorescent markers to image a mouse’s entire body. This revolutionary technique provides detailed 3D maps that will enable a better understanding of biological systems and diseases. Their findings were published in Nature Biotechnology.
More than a century of dedicated work has provided a detailed understanding of the gross anatomy of the human body and the body of common model organisms and has produced detailed histological maps of many individual organs. However, it remains challenging to map the distribution, connectivity and molecular makeup of cell types across the whole body for a given experimental condition. For instance, although the nervous system pervades every region of the mammalian body, cellular-level maps depicting the intricate web of nerves linking various organs and connecting them to the central nervous system are still lacking. In addition, most methods to image nerves or other cells in the context of whole bodies rely on transgenic animals, which limits the flexibility of experimental design. Generating transgenic animals to map changes in the distribution of relevant proteins is usually prohibitively expensive and time consuming. However, such whole-body connectivity maps will be needed to understand the functional interdependence of organ systems and how a disease starting in one part of the body affects distal organs and tissues, for example, during neurodegeneration or systemic inflammation.
Recent clearing methods have enabled labeling and imaging of intact tissues, mouse organs and bodies, parts of human organs and even human embryos. While previous methods, such as CUBIC, PACT, PEGASOS and uDISCO, enabled whole-body imaging, they relied on transgenic expression of fluorescent proteins in a subset of cells, such as mice expressing Thy-1 enhanced green fluorescent protein (EGFP) in neurons. For example, vDISCO relies on the small antibody fragments called nanobodies (roughly one-tenth of IgG size) for whole mouse body labeling. Despite the thousands of conventional antibodies that have been developed over the past decades, only a handful of nanobodies are available or function effectively in a histological context. Consequently, we still lack appropriate, universally applicable labeling methods for whole mouse bodies that use standard IgG antibodies.
Although homogeneous labeling of whole bodies with small molecules (for example, DNA-labeling dyes) or nanobodies can be achieved by cardiac pumping of solutions through the mouse vasculature, standard IgG antibodies face several challenges. These include degradation or precipitation during perfusion, inability to consistently penetrate various tissue layers—including muscles and bones—and insufficient membrane permeabilization to reach deep into all tissues with diverse properties.
To overcome these challenges, we developed wildDISCO (immunolabeling of wildtype mice and DISCO clearing), a chemical method enhancing the penetration of standard (roughly 150 kDa in size) antibodies into the whole roughly 2 cm-thick mouse body. Our method relies on cholesterol extraction for permeabilization to ensure homogeneous penetration and staining across the tissues of the whole mouse body including muscles, bones, the brain and the spinal cord. Combining whole-body antibody labeling with DISCO-based tissue clearing allowed us to provide body-wide maps of cell-type and protein distribution with unprecedented ease and will help advance our understanding of biological systems.
Continue reading this Nature Biotechnology publication: Whole-body cellular mapping in mouse using standard IgG antibodies
Feature image: © HelmholtzMunich | © Ertürk Lab
