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bioRxiv · 10.64898/2026.04.03.716422

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

Kim, C.·Choe, S.-K.·Kim, S.-H.
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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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Kim, C., Choe, S.-K., Kim, S.-H.. 2026-04-08. 3-Minute Hematoxylin and Oil Red O (H-ORO) Staining Protocol for Frozen Sections of Zebrafish. https://doi.org/10.64898/2026.04.03.716422

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