ABSTRACT
Deep immunohistochemistry (IHC) is a nascent field in three-dimensional (3D) histology that seeks to achieve thorough, homogeneous, and specific staining of intact tissues for visualization of microscopic architectures and molecular compositions at large spatial scales. Despite the tremendous potential of deep IHC in revealing molecule-structure-function relationships in biology and establishing diagnostic and prognostic features for pathological samples in clinical practice, the complexities and variations in methodologies may hinder its use by interested users. We provide a unified framework of deep immunostaining techniques by discussing the theoretical considerations of the physicochemical processes involved, summarizing the principles applied in contemporary methods, advocating a standardized benchmarking scheme, and highlighting unaddressed issues and future directions. By providing the essential information to guide investigators in customizing immunolabeling pipelines, we also seek to facilitate the adoption of deep IHC for researchers to address a wide range of research questions.
