Immunofluorescence and Immunohistochemistry

by Peter Delves, PhD

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    In the direct immunofluorescence test, there is a biopsy section or other tissue section placed on a slide. A fluorescein-labeled sheep antibody against the antigen of interest is added. So for example, one might want to see whether a particular patient in a biopsy was expressing a particular tumor antigen. And one could use a sheep antibody specific for that tumor antigen. Under ultraviolet light, the fluorescein label emits a visible light over the antigen of interest. So for example, if we are looking for a specific tumor antigen, then if the patient expresses that tumor antigen, there will be light emission. If they do not, there will be no light emission. In the indirect immunofluorescence test, the tissue can be of animal or human origin as long as it contains the antigen that one is interested in. Patient serum is added, and in this particular example where we are looking for autoantibodies, the autoantibodies bind to the antigen. So this could be a tissue section of thyroid for example, and we’re asking the question: does the patient have autoantibodies to thyroid antigens? A fluorescein-labeled sheep anti-human IgG is added and this binds to the patient’s immunoglobulin gene. The bound IgG is detected under ultraviolet light. One example of this would also be the measurement of anti-nuclear antibodies in patients with systemic lupus erythematosus. Here we have patient’s serum that is incubated with a section of rat liver, a section of rat kidney and a section of rat stomach. Binding to the antigens present in these tissues is detected by a fluorescein labeled anti-human immunoglobulin antibody. And we can see the patient is indeed positive for these anti-nuclear antibodies where we can see fluorescence of a liver section, of a kidney section and of a stomach section. As well as...

    About the Lecture

    The lecture Immunofluorescence and Immunohistochemistry by Peter Delves, PhD is from the course Immunodiagnostics. It contains the following chapters:

    • Direct Immunofluorescence
    • Indirect Immunofluorescence
    • Direct Immunohistochemistry
    • Indirect Immunohistochemistry

    Included Quiz Questions

    1. A secondary antibody
    2. An enzyme
    3. A substrate
    4. A tissue section
    5. Antigen detection
    1. Fluorescein-labeled sheep antibody
    2. HPR
    3. A tissue section
    4. Enzyme substrate
    5. A secondary antibody
    1. An enzyme-labeled antibody and a color change mediating enzyme
    2. Fluorescein-labeled sheep antibody
    3. A secondary antibody
    4. A patient antibody
    5. HPR

    Author of lecture Immunofluorescence and Immunohistochemistry

     Peter Delves, PhD

    Peter Delves, PhD

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