Immunohistochemical Staining Protocol of Enteric Glial Cells in Myenteric Plexi

Despite the development of several genetic approaches to visualize enteric glia within the enteric nervous system (ENS), multiple fluorescence labeling remains an essential tool for simultaneously identifying several target molecules. Fluorescence microscopy is widely used to differentiate between distinct glial subtypes based on their morphological appearance and chemical content, with a high degree of specificity. The dynamic nature of enteric glial cells drives extensive changes in morphology and/or the expression of markers, such as GFAP and S100β, in response to extracellular cues and under distinct physiopathological conditions. These changes depend on factors such as the type and severity of the insult, the time elapsed following the insult, the type of enteric glia, their location within the intestine, and the specific molecular signals they receive. In this context, immunofluorescence provides a combinatorial code that defines the extent of phenotypical changes in distinct glial subtypes based on their protein expression. In this chapter, we describe a protocol designed to immunolabel enteric glia in whole-mount circular muscle myenteric plexus (CMMP) preparations and provide an overview of the main sensitive steps that may affect the quality of the immunolabeling.