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Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry
Published on: December 13, 2013
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Optimization of immunostaining on flat-mounted human corneas.
Fabien Forest1, Gilles Thuret2, Philippe Gain3
1"Corneal Graft Biology, Engineering and Imaging" Laboratory, EA2521, SFR 143, Faculty of Medicine, Jean Monnet University, Saint-Etienne, France; Department of Pathology, University Hospital of Saint-Etienne, France.
Molecular Vision
|January 21, 2016
Summary
Optimizing immunostaining on flatmounted corneas by controlling fixative temperature enhances protein localization and allows detection of rare cells. This refined technique offers significant advantages over traditional methods for studying corneal tissues.
Area of Science:
- Ophthalmology
- Cell Biology
- Biotechnology
Background:
- Conventional immunohistochemistry (IHC) on corneal cross-sections offers limited visualization of corneal endothelial cells (ECs) and lacks precise subcellular localization.
- Previous work established protocols for flatmounted cornea immunostaining using paraformaldehyde (PFA) or methanol fixation.
Purpose of the Study:
- To systematically assess the impact of fixative temperature on immunostaining quality in flatmounted corneas.
- To demonstrate the advantages of optimized flatmounted cornea immunostaining for detecting rare cells and achieving accurate subcellular protein localization.
Main Methods:
- Analyzed staining of ubiquitous proteins (ZO-1, hnRNP L, actin, histone H3) in organ-cultured human corneas fixed with PFA or methanol at various temperatures (4°C, -20°C, 23°C, 37°C, 50°C).
- Applied optimized protocols to fresh corneas for identifying KI67 in Fuch's endothelial corneal dystrophy (FECD) endothelium, localizing neural cell adhesion molecules (NCAMs) in ECs, and characterizing cytokeratin K3/12, CD44, and S100b in corneal epithelium.
Main Results:
- Fixative temperature significantly impacts immunostaining quality; room temperature is recommended over conventional cold fixation for PFA and methanol.
- Optimized protocols revealed proliferating ECs in FECD, suggesting impaired compensatory mechanisms, and precisely localized NCAMs to the lateral EC membranes.
- Demonstrated clear subcellular localization of epithelial markers (K3/12, CD44) and identified S100b-expressing epithelial Langerhans cells.
Conclusions:
- Fixative temperature is a critical parameter for optimizing immunostaining on flatmounted intact corneas.
- Flatmounted cornea immunostaining provides superior whole-tissue overview and subcellular detail compared to cross-section IHC.
- The technique is effective for both corneal endothelium and epithelium, with potential applications for other superficial epithelia.
