Atto 643 Carboxy Selectively Labels Astrocytes with Minimal Oligodendrocyte Cross-Reactivity

Astrocytes are critical regulators of brain homeostasis and circuit function, and sulforhodamine 101 (SR101) are widely used for astrocyte labeling. However, SR101 also labels a significant portion of oligodendrocytes. Here, we identified Atto 643 carboxy, a far-red fluorescent dye, as a highly specific marker for astrocytes with minimal oligodendrocyte labeling. Furthermore, Atto 643 carboxy demonstrates minimal nonspecific uptake in myelin sheaths, and hippocampal pyramidal neuron as reported for SR101. We demonstrate robust imaging of astrocyte morphology and distribution in vivo and in acute brain slices. Our findings establish Atto 643 carboxy as a highly specific small-molecule probe for astrocyte-selective imaging, offering a powerful tool for dissecting astrocyte structure, dynamics, and function in intact brain tissue.