Super-resolution Analysis of Astrocyte Morphology Using Expansion Microscopy

Analyzing the structure of astrocytes, their specific morphological features, and their remodeling is important for understanding how this cell type fulfils its many functions. This is because cell shape determines the propagation of intracellular signals and their subcellular compartmentalization. At the same time, it determines which other cells in the neuropil an astrocyte is closely in contact with and can most effectively exchange signals with. One experimental challenge has been that the most abundant small astrocytic processes cannot be resolved with diffraction-limited microscopy. Typically, this obstacle was overcome by using electron microscopy, but the continuous development of super-resolution microscopy has produced many alternative techniques. One is expansion microscopy (ExM) [1]. ExM, in principle, expands the tissue while preserving the relative positioning of labels that mark structures of interest (e.g., fluorescent labels), which increases the effective spatial resolution of light microscopy without improving the spatial resolution of the microscope itself. The advantage of ExM is that it requires only a little more laboratory infrastructure than immunohistochemistry combined with confocal fluorescence microscopy. We have previously applied this universal technique to the analysis of the structure of astrocytes and of their fine processes and their perisynaptic arrangement. Here, we present a comprehensive protocol for visualizing and localizing astrocytes, synaptic structures, and synaptic and astrocytic proteins in fixed brain tissue.