Immunohistochemistry

When the antigen is cell-associated, immunohistochemistry allows the detection and characterization of antibodies. For tissue sections, immunofluorescence is favored in many laboratories because of its simplicity. It is particularly useful for the detection of antibodies, including autoantibodies, recognizing antigens in intracellular organelles or where double labeling with two different antibodies (detected using two dyes of different color) is advantageous. Fluorescein and rhodamine are the standard conjugates, but a wide range of different colored dyes are available. Laser-based confocal microscopes have greatly increased the sensitivity and specificity of these techniques.

Immunofluorescent techniques for the detection of antibodies are widely used in conjunction with the flow cytometer or fluorescence-activated cell sorter. CD numbers describe leukocyte cell surface antigens recognized by monoclonal antibodies, defined initially by their cytofluorometric staining patterns on a large panel of different cell types. A wide range of different laser dyes are now available which are designed for use with the lasers commonly used in flow cytometers and confocal microscopes.

For tissue immunohistochemistry, enzyme-labeled antibodies are the choice in most laboratories. Peroxidase- and alkaline phosphatase-based techniques are used especially where the antigen detected will withstand the harsh fixation techniques often necessary to maintain tissue integrity. At the electron microscope level, immunogold techniques are used. Small, specifically sized gold particles are attached to antibody to allow localization of antigens. Colloidal gold conjugates can also be used successfully at the light microscope level, particularly in association with silver enhancement techniques. Immunogold can also be applied to blotting techniques giving sensitivity similar to that of enzyme-based detection systems.

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