Deep Insights Without Clearing: An Optimized Protocol for Patient-Derived Whole-Organoid Immunofluorescence Imaging.

Here, we present an optimized whole-organoid immunofluorescence staining protocol that preserves morphology, achieves uniform antibody penetration, and enables deep-tissue imaging without the need for tissue clearing. Organoids are three-dimensional, self-organizing structures that replicate many of the cellular and architectural features of their tissue of origin, making them powerful preclinical models for studying development, disease, and therapeutic responses. Most organoid research is performed on formalin-fixed, paraffin-embedded (FFPE) samples, in which organoids are sectioned across the Z-planes. While this method facilitates conventional histological analysis, it limits spatially resolved visualization of protein expression and tissue organization. The inherent density and abundant extracellular matrix of organoids present substantial barriers to reagent penetration and imaging depth, further constraining the ability to capture their three-dimensional architecture in full. Our new workflow includes steps for fixation, permeabilization, and antibody incubation and has been validated on intestinal organoids using markers for different membrane-associated proteins. By providing high-quality staining through the full organoid depth without clearing, this protocol streamlines sample preparation, reduces processing artifacts, and allows more rapid, accurate 3D mapping of cell types and structures in organoid-based research.