Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment.
Keywords: 3D reconstruction; Immunolocalization; Protein–protein interaction; Tissue multi-protein expression; Whole-mount.