A nuclear fluorescent dye identifies pericytes at the neurovascular unit

Perivascular pericytes are key regulators of the blood–brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, pericyte identification remains challenging. A previous study reported that the far‐red fluorophore TO‐PRO‐3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO‐PRO‐3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO‐PRO‐3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron‐glial antigen 2 (NG2) and platelet‐derived growth factor receptor beta antigen (PDGFrβ) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO‐PRO‐3 signal enabled quantification of pericytes density and morphometry; likewise, TO‐PRO‐3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO‐PRO‐3 stained cells expressed the contractile protein α–SMA, indicative of their ability to control the capillary diameter. Uptake of TO‐PRO‐3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein‐mediated active transport sustained dye incorporation. We conclude that TO‐PRO‐3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature. Identification of capillary pericytes remains challenging. The nuclear far‐red fluorophore TO‐PRO‐3 was previously employed to label the pericytes of the subventricular zone (Lacar et al., 2012). Herein, we show that ex vivo living pericytes from the hippocampus, cortex, spinal cord, and retina selectively uptake TO‐PRO‐3. Uptake involves a transporter that actively concentrates the dye into pericytes allowing their identification. The good stability, brightness, affordability, and reproducibility of the fluorescent signal, together with the ease of the labeling procedure, makes TO‐PRO‐3 a convenient method to trace cerebral pericytes during multiple labeling circumventing the need for reporter mice or immunohistochemical counter‐staining.