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Department of Molecular Physiology

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fluo-microscopy
Immunohistochemistry



The first step in the examination of the role of distinct proteins in transduction processes is to identify the proteins and to localize their site of expression. Both can be done using antibodies which carry a fluorescent label. The principle of this technique (“immunohistochemistry”) is illustrated on the right. Thin tissue sections or cultivated cells are fixed on a glass support and are permeabilized to allow antibodies to enter the cell. A solution containing antibodies directed specifically against one target protein (or two different antibodies against two different target proteins) is then placed on top of the sample. A thorough rinse with buffer solution then removes all antibodies which are not bound to their target protein. In a second step, labeled antibodies are added which bind to the first antibodies and make them visible. Since several labeled antibodies can attach to each first antibody, an optical signal becomes visible in the microscope wherever the target protein is expressed.

The images above, left show fluorescence originating from cultivated cells. The green light is emitted by a protein (GFP = green fluorescent protein) expressed by these cells. The red light comes from a second antibody which recognizes an antibody against a test protein that was experimentally expressed in these cells to study its properties.

The images on the right show a cross section (a coronal section) of the sensory surface of the olfactory organ within the nose. Blue light originates from cell nuclei (DAPI-stain) where as the  red line at the surface indicates the expression of the enzyme adenylyl cyclase III (AC III). This enzyme is located in the chemosensory cilia of olfactory neurons at the tissue surface. During odor detection, AC III synthesizes cAMP, the intracellular messenger of the olfactory signal transduction cascade.




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