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Updated: Jun 25, 2026
Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse
Published on: December 22, 2020
A novel protocol of whole mount electro-immunofluorescence staining
Hongshan Liu1, Winston W Y Kao
1Department of Ophthalmology, University of Cincinnati, Cincinnati, OH 45267-0838, USA.
Insights
A novel electro-immunofluorescent staining method enhances reagent penetration in whole mount corneas, improving antigen detection across all corneal layers. This technique offers superior results compared to conventional methods for corneal research.
Area of Science:
- Ophthalmology
- Biotechnology
- Cell Biology
Background:
- Whole mount immunostaining of the cornea is crucial for studying its structure and disease.
- Conventional methods often suffer from poor reagent penetration and high background noise.
- Improved techniques are needed for comprehensive analysis of corneal tissues.
Purpose of the Study:
- To develop a novel whole mount immunostaining technique for the cornea.
- To enhance the penetration of staining reagents into corneal layers.
- To reduce non-specific binding and background signal.
Main Methods:
- Adult mouse corneas were fixed and embedded in agarose.
- Fluorescent staining reagents were applied and electrophoresed using a submarine gel electrophoresis apparatus.
- Conventional immunostaining protocols were used for comparison.
- Confocal laser scanning microscopy (CLSM) was employed for evaluation.
Main Results:
- The electro-immunofluorescent staining method demonstrated uniform antigen distribution in corneal stroma, epithelium, and endothelium.
- Specific antigens like keratocan, beta-tubulin, and focal adhesion kinase (FAK) were clearly detected.
- Conventional methods showed limited penetration of IgG conjugates into corneal stroma and epithelium.
- Phalloidin staining revealed similar results to conventional methods for epithelial healing.
Conclusions:
- Whole mount electro-immunofluorescent staining effectively detects antigens in all corneal layers.
- This method overcomes penetration limitations of conventional techniques.
- It provides a valuable tool for corneal research and diagnostics.
Purpose:
To develop a new method of whole mount immunostaining that improves the penetration of staining reagents into the cornea and decreases non-specific binding and background.
Methods:
Adult mouse corneas were fixed overnight in 4% paraformaldehyde or a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4 degrees C. After washing with 0.1% Triton X-100, corneas were embedded in 1% solidified agarose in a plastic column and fluorescent staining reagents, e.g., FITC-IgG (Fluorescein isothiocyanate-immunoglobulinG) conjugates in 0.5% solidified agarose was overlaid onto the specimens. The column was directionally immersed in a submarine gel electrophoresis apparatus filled with Tris-glycine buffer (TGB, pH=7.4) and electrophoresed at 4-10 mA for 10-24 h. For comparison, conventional protocols of immune fluorescent staining were also employed. The outcomes were evaluated by confocal microscopy.
Results:
Antibody conjugates recognizing extracellular matrix (ECM) components, integral membrane protein, and intracellular structural proteins were used in whole mount corneas. The images of confocal laser scanning microscopy (CLSM) displayed a uniform distribution pattern of keratocan in corneal stroma, which is similar to that of section-staining. Anti-beta-tubulin antibodies bound to microtubes that are distributed within the whole cell body of superficial corneal epithelium cells and stromal keratocytes, but it was found perinuclear of corneal epithelial wing layers and endothelium; integral membrane protein, FAK (focal adhesion kinase), specifically labeled stromal cells of keratectomy corneas that healed for three weeks. In comparison, conventional protocols of immune fluorescent staining using the same antibody conjugates were also employed but did not yield satisfactory results. It was found that IgG conjugates examined did not readily penetrate into stroma and/or intact corneal epithelium. Phalloidin is a small molecule that can readily penetrate into deep tissue and preferentially binds to F-actin. After the whole mount electrofluorescent staining of phalloidin-rhodamine in the mouse cornea, the results were the same as conventional whole mount staining during the healing of epithelial debridement. The cytoplasmic protrusion formed by lamellipodia and filopodia can be clearly demonstrated.
Conclusions:
These results indicate that the whole mount electro-immunofluorescent staining allows the detection of antigens in all layers of cornea, i.e., epithelium, stroma, and endothelium.
