Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

Spatial analysis of cells and their microenvironment within tissues enhances our understanding of biological processes. Ideally, a broad range of biomolecules should be analyzed in large 3D tissue specimens at subcellular resolution. Here, we present a protocol to identify and extract target sections from previously cleared tissues. We describe steps for combining 3D light sheet imaging and subsequent 3D-guided deep cell phenotyping via multi-cyclic 2D microscopy.