Abstract

Accurate assessment of blood-brain barrier (BBB) penetration is essential for validating small-molecule fluorescent probes. Conventional methods often suffer from signal attenuation, tissue damage, or fluorescence distortion during chemical fixation. We present an optimized histological workflow: ‘intraperitoneal administration → fresh brain sampling → unfixed cryosectioning → confocal imaging.’ By utilizing fresh-frozen tissue without chemical fixation, this method preserves intrinsic probe fluorescence and provides a high-resolution, accurate representation of BBB penetration. The workflow was validated across three murine models: neonatal hypoxic-ischemic injury, neuroinflammation, and neurodegenerative disease. In all models, fluorescence intensity showed a strong positive trend correlated with pathological severity, demonstrating that the method is exceptionally sensitive for detecting pathology-associated BBB penetration changes. While the workflow preserves baseline signals in control groups, its primary advantage lies in providing a high-resolution, accurate representation of probe engagement in compromised CNS environments. Notably, this standardized approach avoids invasive cranial procedures and minimizes fixation-induced quenching. The primary advantage of this optimized workflow over conventional fixation-based methods is the superior preservation of intrinsic small-molecule fluorescence in fresh-frozen sections, ensuring a more reliable evaluation of BBB penetration in preclinical research.