Robust paraffin embedding of 3D cultured vocal fold epithelial organoids

Vocal fold organoids recapitulate critical structural and functional features of native vocal fold mucosa, providing a physiologically relevant model for investigating vocal fold biology and disease mechanisms. However, conventional histological processing of these organoids remains technically demanding, often resulting in substantial sample loss, inadequate visualization during embedding and difficult retrieval the blocks from Eppendorf tubes. To address these issues, we established a comprehensive protocol that integrates direct eosin pre-staining, agarose pre-embedding and fine-needle-assisted retrieval of agarose-embedded blocks. This optimized workflow increases processing efficiency, enables enhanced visual monitoring and better preserves cytoarchitectural integrity. Consequently, sectioning is facilitated, and both hematoxylin and eosin (HE) staining and immunofluorescence (IF) exhibit superior quality and reproducibility, producing highly consistent morphological details and robust signal resolution. The proposed method provides a standardized and reliable platform for high-resolution histological and IF examination of epithelial organoids, thereby extending its utility in vocal fold research and related organoid applications.