Microglial cells are the immune cells of the CNS. They monitor the integrity and react to any disturbance of brain homeostasis. Even though they play a critical role in many CNS diseases, little is known about functional properties of microglia in vivo. Here we introduce a new protocol for in vivovisualization of microglial cells. It utilizes Tomato lectin (TL), a well-known histological marker of microglia (Acarin, 1994). A brief (30-s-long) pressure injection of TL conjugated with a red fluorescent dye DyLight^® 594 (25 mg/ml in the injection micropipette, 40 kPa) into the brain tissue resulted in a robust staining of microglial cells in an area with the diameter of ~ 100 mm. This protocol was applicable to animals of any age (2–23 month) as well as to mouse models of Alzheimer's disease. It enabled visualization of an entire cell at high spatial resolution, comparable to that obtained when imaging eGFP-expressing microglia. Similar staining quality was obtained with TL conjugated with a green dye fluorescein. The quality of TL-based staining was preserved in fixed tissue processed for post-hoc immunocytochemistry. Importantly, TL-based staining did not modify morphological and functional properties of microglia. TL-labeled microglia extended their processes towards the ATP source at the same speed as control cells did and similarly responded to the activation of ionotropic and metabotropic P2 receptors. Furthermore, TL did not modify spontaneous activity of the surrounding neuronal network. Thus, the protocol described in this study represents a versatile tool forin vivo studies of microglia.