An Integrated Platform for the Comprehensive Analysis of Host-Fungal Interactions in the Murine Tongue

This protocol describes a mouse model of Candida albicans oral colonization that establishes stable, long-term fungal persistence in the oral cavity, thereby recapitulating key features of human commensal colonization, including the induction of sustained adaptive antifungal immunity. The model enables the analysis of host-fungal interactions across multiple experimental readouts. Procedures are provided for isolation of single-cell suspensions from the whole tongue, the tongue epithelium, and the draining lymph nodes for flow cytometric analysis, supporting both enumeration and extended functional characterization of immune cell populations. Antigen-specific T cell responses can be assessed by re-stimulation of isolated T cells with C. albicans-pulsed dendritic cells prior to staining and analysis. Additional methods include quantification of fungal burden in tongue tissue, histological assessment of fungal localization and immune cell infiltration, and isolation of high-quality RNA using an optimized extraction workflow compatible with RT-qPCR and RNA sequencing. Collectively, these protocols provide a flexible and integrated platform for studying immune responses and host-fungal interactions during persistent oral colonization. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Oral colonization of mice with Candida albicans Basic Protocol 2: Preparation of single-cell suspensions from the murine whole tongue, tongue epithelium, and draining lymph nodes Basic Protocol 3: Re-stimulation of cLNs and tongue T cells for qualitative and quantitative assessment of T cell effector functions Basic Protocol 4: Flow-cytometry-based analysis of tongue immune cell populations Support Protocol 1: Evaluation of fungal burden in the tongue of C. albicans-colonized mice Support Protocol 2: Visualization of tissue integrity, tissue inflammation, and fungal localization by histology Support Protocol 3: Extraction of total RNA from the tongue of C. albicans-colonized mice for RNA sequencing and RT-qPCR.