Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin |
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| Authors: | D Hicks R S Molday |
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| Affiliation: | 1. Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany;2. German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany;3. Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK;4. Department of Clinical Genetics, VU University Medical Center, Amsterdam, the Netherlands;5. Centre for Genetic Epidemiology, Institute for Clinical Epidemiology and Applied Biometry, University of Tübingen, Tübingen, Germany;6. Institut du Cerveau et de la Moelle épinière, ICM, Inserm U 1127, CNRS, UMR 7225, Sorbonne Universités, UPMC University Paris 06, UMR S 1127, AP-HP, Pitié-Salpêtrière Hospital, Paris, France;7. Departments of Neurology and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA;8. Movement Disorders Unit, Department of Neurology, Hospital Universitari Mutua de Terrassa, Barcelona, Spain;9. Movement Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, Barcelona, Spain;10. Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), Barcelona, Spain;11. Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain;12. Department of Neurosciences, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, Lisbon, Portugal;13. Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Lisbon, Portugal;14. Department of Epidemiology, Erasmus MC, Rotterdam, the Netherlands;15. Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands;p. Netherlands Consortium for Healthy Ageing (NCHA), Rotterdam, the Netherlands;q. Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, USA;1. Department of Ophthalmology, College of Medicine, King Saud University, Riyadh, Saudi Arabia;2. Rega Institute for Medical Research, Department of Microbiology and Immunology, University of Leuven, KU Leuven, Belgium;1. Department of Surgery, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada;2. Department of Surgery, University of Toronto, Toronto, ON, Canada;3. Department of Anaesthesia, Sunnybrook Health Sciences Centre, Toronto, ON, Canada |
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| Abstract: | Eleven monoclonal antibodies against different segments of bovine rhodopsin were used with immunogold-dextran markers to label Lowicryl thin sections of bovine and frog retinal photoreceptor cells for visualization by transmission electron microscopy. Antibodies against the C-terminus, F1-F2 loop and N-terminus of rhodopsin were all observed to label bovine rod outer segments (ROS) densely, but to label rod inner segments (RIS) only sparsely. Most antibodies bound 200-600 gold particles per micron2 in the ROS, 10-60 gold particles per micron2 in the RIS and 5-20 particles per micron2 on the Lowicryl resin. One antibody against the N-terminus and one antibody against the C-terminus resulted in the binding of over 1000 particles per micron2 in bovine ROS. Cone outer segments (COS) were labeled with only one antibody, rho 3D6, having a specificity for the 1'-4' C-terminus of bovine rhodopsin. Ninety per cent of the COS were observed to be labeled with this antibody. Immunogold-dextran labeling was also used to study the cross-reactivity of these antibodies to rhodopsin in red and green frog ROS and COS. Monoclonal antibodies directed against sites along the F1-F2 loop, and the N-terminus labeled red frog ROS densely, but did not label either green ROS or COS. Three C-terminal specific antibodies against binding sites along the 1'-8' segment labeled both green and red ROS, but a higher extent of labeling was observed on the green ROS. Antibody rho 3D6, which bound to bovine COS, also labeled frog COS. These results indicate that the F1-F2 loop and segments along to the N-terminus and the C-terminus of bovine rhodopsin show a high degree of homology with corresponding regions of frog rhodopsin from red ROS; the C-terminal 1'-8' segment of bovine rhodopsin is closely related to the corresponding segment of frog rhodopsin from green ROS; and the 1'-4' C-terminus of bovine rhodopsin is highly homologous to segments of opsin from most bovine and frog COS. The labeling of frog red ROS in relation to multiple forms of rhodopsin observed by SDS-gel electrophoresis is discussed. |
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