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Related Experiment Video

Updated: Jul 26, 2025

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches
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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.

Current Protocols
|June 20, 2023
PubMed
Summary
This summary is machine-generated.

This study presents a whole-mount skin staining protocol for 3D imaging of immune cells and structures. The method allows detailed analysis of skin

Keywords:
FIJINeighborhood Frequency and Normalized Median Evenness (NME)Spatial Distribution Index (SDI)cell profilerconfocal laser scanning microscopy (CLSM)fluorescence-conjugated primary antibodyimage analysispolychromatic immunofluorescent stainingwhole-mount skin

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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.