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

Kim, C.; Choe, S.-K.; Kim, S.-H.

2026-04-08 pathology
10.64898/2026.04.03.716422 bioRxiv
Show 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. Mayers 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.

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