3-Minute Hematoxylin and Oil Red O (H-ORO) Staining Protocol for Frozen Sections of Zebrafish

Abstract Optimized histological techniques are crucial for visualizing cellular morphology across zebrafish tissues. Here, we report a rapid and reliable hematoxylin and Oil Red O (H-ORO) staining protocol for frozen sections that can be completed in less than three minutes. Mayer’s hematoxylin is used for nuclear staining, followed by Oil Red O (ORO) to visualize lipid-rich structures such as the endomysium surrounding myofibers, white matter of the brain, and myelin layers of major axonal tracts. Importantly, our optimized H-ORO protocol preserves tissue integrity and minimizes artifacts such as myofiber shrinkage commonly observed with ethanol-based hematoxylin and eosin (H&E) staining in both frozen and paraffin sections. Competing Interest Statement The authors have declared no competing interest.