In this chapter, we describe the technical details to visualize and analyze effector immunological synapses between T cells and astrocytes in the brain with high-resolution confocal imaging. This procedure is critical for the optimal and even penetration of labeling antibodies within the nerve tissue to obtain accurate staining and allow a uniform three-dimensional analysis of the T cell-astrocyte interactions. We emphasize here the comprehensive exploration of the tissue and analysis with confocal microscope as well as the display of microanatomical details of the three-dimensional reconstruction for interface visualization (including peripheral and central supramolecular activation clusters, effector molecules, and other organelles such as microtubule organizing centers (MTOCs) and Golgi apparatus).
Keywords: 3D reconstruction; Astrocyte; Confocal microscopy; Immunofluorescence; T cell; Tissue; c-SMAC; p-SMAC.