Vocal fold mucus layer: Comparison of histological protocols for visualization in mice.

Objectives: The epithelial associated mucus layer of vocal fold (VF) mucosa, plays an essential role in protecting and lubricating the tissue, as well as promoting normal voice quality. Serving as a habitat for laryngeal microbiota involved in the regulation of host immunity, VF mucus contributes to laryngeal health and disease. However, its unstable structure renders its' investigation challenging. We aim to establish a reproducible histological protocol to recover the natural appearance of the VF mucus layer for investigation. Methods: Using a murine model, we compared the suitability of multiple fixation methods-methacarn, formalin, and cryopreservation followed by post-fixation with formalin, paraformaldehyde (PFA), acetone, and two staining methods-Alcian Blue (pH 2.5)/Periodic Acid Schiff (AB/PAS) or PAS. Fixation and staining outcomes were evaluated based on the preservation of tissue morphology and mucus layer integrity. Mucin proteins, Muc1 and Muc4, were stained to validate the presence of mucus layer overlaying the VF mucosa. Results: Methacarn fixation followed by PAS staining was capable of preserving and displaying the smooth and continuous mucus layer, ensuring the determination of mucus thickness and mucin staining. Conclusions: Our study if the first to establish a histological protocol for the visualization of the in situ VF mucus layer whereby facilitating the study of VF mucus biology including VF surface hydration, ion/nutrients transports, biomechanical properties that maintains normal voice quality as well as VF pathophysiology and host-microbe interactions in the larynx. Level of Evidence: N/A.