Home › Microscopy & Imaging › Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

DOI: 10.3791/56106-v
What you'll learn
  • ✓ Prepare and cryosection thick skin tissue samples for three-dimensional analysis
  • ✓ Apply immunofluorescent labeling while preserving antigenicity in whole mount preparations
  • ✓ Mount and image horizontal tissue cross-sections using confocal microscopy
  • ✓ Compare advantages of horizontal whole mount versus classical cryosection approaches
Protocol

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Difficulty
intermediate
Total time
~4–6 hours per sample (including cryosectioning, immunolabeling, and mounting)

Steps

1
Prepare skin tissue samples for processing

Obtain and prepare skin tissue specimens for subsequent cryosectioning. Ensure samples are appropriately handled to preserve tissue integrity and antigenicity.

▶ 00:55
2
Cryosection tissue into thick horizontal sections

Cut thick, three-dimensional horizontal cross-sections of skin using cryosectioning techniques. This preserves tissue structure and enables full confocal imaging depth.

▶ 02:07
3
Apply immunofluorescent labeling to sections

Perform immunofluorescent staining on mounted tissue sections using primary and secondary antibodies. This step preserves antigenicity across the thick tissue volume.

▶ 03:17
4
Mount labeled sections for microscopic visualization

Prepare and mount immunolabeled tissue sections on microscope slides using appropriate mounting media. Optimize mounting to enable high-quality confocal imaging.

▶ 05:05
5
Compare horizontal whole mount with classical cryosection

Image and evaluate horizontal whole mount preparations versus conventional cryosections. Assess improvements in tissue preservation, three-dimensional visualization, and histological detail.

▶ 06:36
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