Evolution of visual pigment and differentiation of membrane structure : Production of monoclonal antibody and electron microscopic immnunohistochemistry

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1.Production of monoclonal antibodies against lamprey-retinal homogenate.We have developed 10 new monoclonal antibodies. Their binding sites were detected on the sectioned retina by optical and electron microscopic immunohistochemistry. Molecular weight of antigens were estimate by western blotting.1) W26, W27, W39 : Specifically bound to the outer segment of short photoreceptor cell (SPC).2) W226, W231, W312, W425 : To the outer segment, inner segment except for ellipsoid of SPC and axon.3) W15 : To the outer and inner segment of SPC.4) W38 : To the all region of SPC.5) W318 : To the neural layr of retina.Following 3 monoclonal antibodies were specialized for a molecular component involved in photoreceiving procedure.1) W39 : For lamprey rhodopsin, molecular weight is about 40kDa.2) W312, W425 : For lamprey arrestin, molecular weight is about 49kDa.2.Establishment of quantitative immunogold electron microscopy.The specimen was fixed with 4% paraformaldehyde and embedded in LR-white. There were considerably remakable washing out and trace of destruction in the neural layr of retina, and deformation of outer segment of photoreceptor cells as compared with conventional fixation. As far as photoreceptor cells are concerned, however, fortuitously preserved outer segments were found as assosiated variously with fine gold particles, allowing our quantitative examinatoin.ConculusionA series of anti-lamprey retinal antibodries were produced and used for recognition of protein, glycoconjugate and lipids in retinal cells. These antibodies are apparently useful as probes for relationship between corresponding cells of different species.