Expansion Microscopy of the Enteric Nervous System: A Feasibility Study

Expansion microscopy (ExM) enables conventional light microscopes to achieve nanoscale resolution by physically enlarging biological specimens. While ExM has been widely applied in neurobiology, it has not been adapted for the enteric nervous system (ENS). Here, we provide a detailed and reproducible protocol for applying ExM to mouse colonic ENS tissue. The procedure includes preparation of the external muscle layers with the myenteric plexus, histochemical staining for NADPH-diaphorase, immunostaining for glial fibrillary acidic protein (GFAP), anchoring of biomolecules, gelation, proteinase K digestion, and isotropic expansion in a swellable polymer matrix. Step-by-step instructions, required reagents, and critical parameters are described to ensure robustness and reproducibility. Using this protocol, tissues expand 3-5-fold, allowing neuronal somata, fibers, and glial cell processes to be clearly visualized by standard brightfield or fluorescence microscopy. The tissue architecture is preserved, with distortion in the X-Y plane of about 7%. This protocol provides a reliable framework for high-resolution structural analysis of the ENS and can be readily adapted to other peripheral tissues.