Immunohistochemical Localization and Anatomical Distribution of Enteric Glia Cells in Enteric Nervous System

The enteric nervous system (ENS) comprises highly heterogeneous glia cell populations, which play critical roles in the regulation of intestinal neuromuscular function, providing physical and immune protection to neuronal cells and processes in physiological and pathological conditions. Due to the high muscle/mucosa content, thinness, and softness, freezing and sectioning of intestinal tissue are difficult, and distortion of tissue morphology might occur. However, whole-mount immunostaining for glia characterization in the 3D environment of the ENS is a better choice, as this powerful technique facilitates the visualization of different cell populations and their interaction with other cells within the neuromuscular compartments of the intestinal wall, without tissue distortion or damage. In the last decades, novel methodologies for whole-mount staining have been described to preserve the structural integrity of all the neuroglial cell units across the intestinal wall, allowing high-resolution image acquisition. In this chapter, we describe a step-by-step protocol to perform immunofluorescence staining of enteric glial cells in both the myenteric and submucosal plexuses of different regions of the gastrointestinal tract and to optimally visualize cellular constituents of the ENS with confocal microscopy.