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Q. Zeng, E. Lato, S. Schaal, T. H. Tezel; Human Retinal Pigment Epithelium (RPE) Immunoglobulin Synthesis can Puzzle the Interpretation of RPE-Immunostaining. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5934.
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To determine the validity of RPE immunostaining in the view of recent discovery of local immunoglobulin production in human RPE.
Three human donor eyes (65-73 years old) were obtained from the eye bank and fixed in 4% paraformaldehyde at 4°C for 24 hours. 10x10 mm blocks of the eye wall were embedded in paraffin and serially sectioned at 10 µm. Sections were blocked using 3% bovine serum albumin in PBS with 1% Triton for 30 min at room temperature. Sections were then incubated with equimolar amounts of fluorescein isothiocyanate-conjugated anti- human, goat, rabbit and mouse anti-immunoglobulin G. Twelve different sections were studied with fluorescein-microscope and images were obtained at 20 x using same exposure settings. Digital images were studied morphometrically to determine the amount of fluorescent signal generated over RPE with each secondary antibody. Contribution of RPE autofluorescence to the results RPE was calculated using unstained sections.
Incubation with secondary antibodies increased the amount of fluorescent signal originating from human RPE significantly (p<0.05) in the order of human (97.3) >mouse (22.3) > rabbit (9.3) >goat (4.8). Increased fluorescence was a result of increase in both signal area and intensity.
Immunoglobulin production in human RPE may result in increased fluorescence and thus decreased signal-to-noise ratio. Staining with primary antibodies tagged with fluorescent probes or using secondary antibodies that has less cross-reactivity may alleviate this problem.
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