Color Image Analysis of Lymphocyte Subsets in Frozen Section in Experimental Uveitis

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We have tried to elucidate the dynamic changes of lymphocyte subsets in experimental uveoretinitis induced by S-antigen (S-Ag).1. Isolation of bovine S-Ag: Soluble fraction of crude bovine retinas was collected by hypotonic processing and submitted salt fractionation by saturated ammonium sulfate. After the removal of insolubel material by centrifugation the clear supernatant solution was applied to a Sephadex G-200 and to a SDS gel electrophoresis. After condensation by a millipore filter, the S-Ag fraction was purified by adsorption chromatography with stepwise increases in phosphate molarity.2. Production of experimental uveoretinitis in rats by retinal S-Ag: Lewis rats were immunized with 50 micrograms of S-Ag with complete Freund's adjuvant injected into the hind footpads. Uveitis was produced 12-18 days after immunization. Eyes were enucleated and processed immediately after immunization, and on Days 1, 2, 3, 7, 14, 21, and 28.3. Staining of lymphocyte subsets by monoclonal antibodies and immunoperoxidase method: After blocking process by normal gout serum, frozen sections were reacted with mouse hybridoma-derived monoclonal antibokies against four kinds of rat lymphocytes, with peroxidaselabeled goat anti-mouse IgG, and with the substrate solution.4. Color image analysis: The developed program detects IHP (I:intensity,H:hue, P:purity) of a color vector and makes the hue the main index of identification, in order to identify, to binarize, and to measure the red-brown, the color of the substrate reacting on peroxidase. The program also controls an autostage of a microscope, scanning labels in all fields without omission or duplication. The volume (s) of a kind of antigens on the surface of lymphocytes is expressed as an area rate (s/S) to the area of the background tissue (S).