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Skin Whole-Mount Immunofluorescent Staining Protocol, 3D Visualization, and Spatial Image Analysis
Alfonso J Schmidt1,2, Graham D Wright3, Franca Ronchese2
1Hugh Green Cytometry Centre, Wellington, New Zealand.
Insights
This study presents a whole-mount skin staining protocol for 3D imaging of immune cells and structures. The method allows detailed analysis of skin
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Whole-mount skin staining offers a 3D view of skin structures and immune cells, bypassing traditional sectioning.
- Understanding skin's immune defenses against pathogens requires detailed characterization of resident cell types and their spatial organization.
Purpose of the Study:
- To provide a detailed protocol for polychromatic immunofluorescent staining of whole-mount mouse skin.
- To enable visualization and quantitative analysis of immune cell populations and tissue architecture in three dimensions.
- To facilitate the study of skin's immunological strategies in combating pathogens.
Main Methods:
- Polychromatic immunofluorescent staining of whole-mount mouse skin using antibodies against structural markers (CD31, LYVE-1) and immune cells (MHCII, CD64, CD103, CD326).
- Confocal laser scanning microscopy (CLSM) for high-resolution 3D imaging.
- Image processing and visualization using open-source software (ImageJ/FIJI) including z-projections, orthogonal views, 3D rendering, and animation.
- Quantitative spatial analysis using CellProfiler to assess cell-type relationships with indices like Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME).
Main Results:
- An optimized staining panel successfully visualizes blood vessels, lymphatic networks, antigen-presenting cells, macrophages, monocytes, dendritic epidermal T cells, and Langerhans cells.
- Established pipelines for 3D image visualization and quantitative spatial analysis of cell distribution and interactions.
- Demonstrated feasibility of using commercially available reagents and freely available software for comprehensive skin immune cell analysis.
Conclusions:
- The presented protocols enable robust staining, imaging, and analysis of whole-mount mouse skin.
- This methodology enhances the understanding of skin's structural and immunological landscape.
- Researchers can now effectively study skin's defense mechanisms using accessible tools and techniques.
Abstract:
The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.
