Spatial analysis of tissue immunity and vascularity by light sheet fluorescence microscopy

The pathogenesis of cancer and cardiovascular diseases is subjected to spatiotemporal regulation by the tissue microenvironment. Multiplex visualization of the microenvironmental components, including immune cells, vasculature and tissue hypoxia, provides critical information underlying the disease progression and therapy resistance, which is often limited by imaging depth and resolution in large-volume tissues. To this end, light sheet fluorescence microscopy, following tissue clarification and immunostaining, may generate three-dimensional high-resolution images at a whole-organ level. Here we provide a detailed description of light sheet fluorescence microscopy imaging analysis of immune cell composition, vascularization, tissue perfusion and hypoxia in mouse normal brains and hearts, as well as brain tumors. We describe a procedure for visualizing tissue vascularization, perfusion and hypoxia with a transgenic vascular labeling system. We provide the procedures for tissue collection, tissue semi-clearing and immunostaining. We further describe standard methods for analyzing tissue immunity and vascularity. We anticipate that this method will facilitate the spatial illustration of structure and function of the tissue microenvironmental components in cancer and cardiovascular diseases. The procedure requires 1–2 weeks and can be performed by users with expertise in general molecular biology.