Efficient RNA and RNA-protein co-detection in 3D colonoids by whole-mount staining

Area of Science:

• Cell Biology
• Molecular Biology
• Histology

Background:

• Accurate visualization of RNA and proteins within complex 3D tissue structures is crucial for understanding cellular function.
• Existing methods may not adequately preserve tissue architecture or allow for simultaneous detection of both nucleic acids and proteins.

Purpose of the Study:

• To describe a refined protocol for combined fluorescence in situ hybridization (FISH) and immunofluorescence.
• To enable independent or simultaneous visualization of RNA oligos and proteins within human colonoids.
• To adapt the workflow for 3D organoid models from various tissues and organisms.

Main Methods:

• Whole-mount staining technique to maintain colonoid integrity.
• Fixation and mounting procedures optimized for glass slides.
• Integration of FISH for RNA detection and immunofluorescence for protein detection.

Main Results:

• Successful visualization of RNA and proteins within intact human colonoid structures.
• Demonstration of independent and combined imaging capabilities.
• Protocol adaptability for diverse 3D organoid systems.

Conclusions:

• The described protocol offers a robust method for multiplexed molecular analysis in 3D organoids.
• This technique enhances the study of spatial gene and protein expression in complex biological systems.
• The workflow provides a valuable tool for researchers in developmental biology and disease modeling.