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H.N. Liu, W.W. Y. Kao; Whole Mount Electro–Immunohistochemistry . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5047.
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Regular Immunohistochemistry is a useful technique in evaluating the expression pattern of gene products in normal and diseased tissues. However, the technique often faces some difficulty in detecting antigen whose distribution is restricted to a small number of cells in tissue. To overcome this limitation, we have developed a whole mount electro–immunohistochemical method.
Mouse corneas (from Wild type, Kera–/– and Lum–/– mice) were fixed in 4% paraformaldehyde or mixture of 4% paraformaldehyde and 0.1% glutaraldehyde at 4°C overnight. The cornea was immersed in 5X tris–glycine (TGB) buffer to block excess aldehyde for 1 h and permeated with 0.1% triton X–100 in 1X tris–glycine buffer. The tissue was embedded in an electrophoresis column with 1% agarose, 0.05% triton X–100 in 1 X TGB. The tissue was over layered with antibody–Alexa conjugates in 0.5% agarose and 2% agarose in TGB. The antibody–Alexa conjugate was electrophoresed into the tissue based on the electric charge of the antibody in the buffer (pH, 7.4). Electrophoresis was carried out at 10 mA/7–8 voltages for 20–24 hours. After electrophoresis, the cornea was flat mounted and examined by fluorescent microscopy and/or confocal microscopy.
The images of confocal microscopy and fluorescent microscopy showed that both goat anti–keratocan Alexa555 conjugate and goat anti–lumican Alexa488 conjugate specifically labeled corneal stroma with keratocan mostly in the anterior portion of stroma whereas lumican was at the posterior stroma. No keratocan and lumican were detected in corneas of Kera–/– and Lum–/– mice by anti–Kera and anti–Lum antibodies, respectively. Phalloidin–stained cornea revealed that positive cells are throughout corneal epithelium, stroma, and endothelium. In corneal stroma, positive cells are displayed as stellate or dendritic shape and the cytoskeleton are readily apparent. In corneal epithelium and endothelium, the labeling appears as a circumferential band surrounding the cells. After corneal epithelial wound, electro–immunostaining with phalloidin–Rhodamine clearly showed that cells surrounding the wound spread out and extend processes into the wound area.
Electro–immunostaining allows the penetration of antibody molecules into the mouse cornea to label antigens in different cell layers, i.e., epithelium stroma and endothelium with very low non–specific background. Electro–immunohistochemistry is a reliable novel technique for corneal whole–mount immunostaining.
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