Protocol for visualizing microglial lysosomal content by immunohistochemistry in the rodent brain

Microglia, the resident phagocytes of the central nervous system, clear diverse substrates in development, aging, injury, and disease. Here, we present a protocol to visualize phagocytosed content within microglial lysosomes in the rodent brain using immunohistochemistry and confocal microscopy. We describe the steps for tissue staining, image acquisition, and lysosome analysis. We then detail the procedures for microglia and lysosome reconstruction for 3D visualization. For complete details on the use and execution of this protocol, please refer to Gu et al. 1 • Steps for immunohistochemistry and super-resolution imaging of microglial lysosomes • Procedures for lysosome analysis using Fiji/ImageJ • Instructions for microglia and lysosome 3D reconstruction in Imaris Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Microglia, the resident phagocytes of the central nervous system, clear diverse substrates in development, aging, injury, and disease. Here, we present a protocol to visualize phagocytosed content within microglial lysosomes in the rodent brain using immunohistochemistry and confocal microscopy. We describe the steps for tissue staining, image acquisition, and lysosome analysis. We then detail the procedures for microglia and lysosome reconstruction for 3D visualization.