Immunofluorescence and Immunohistochemistry

by Peter Delves, PhD

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    00:01 In the direct immunofluorescence test, there is a biopsy section or other tissue section placed on a slide.

    00:15 A fluorescein-labeled sheep antibody against the antigen of interest is added.

    00:22 So for example, one might want to see whether a particular patient in a biopsy was expressing a particular tumor antigen.

    00:31 And one could use a sheep antibody specific for that tumor antigen.

    00:37 Under ultraviolet light, the fluorescein label emits a visible light over the antigen of interest.

    00:45 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.

    00:54 If they do not, there will be no light emission.

    00:58 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.

    01:10 Patient serum is added, and in this particular example where we are looking for autoantibodies, the autoantibodies bind to the antigen.

    01:21 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.

    01:43 The bound IgG is detected under ultraviolet light.

    01:48 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.

    02:13 Binding to the antigens present in these tissues is detected by a fluorescein labeled anti-human immunoglobulin antibody.

    02:22 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.

    02:35 As well as these immunofluorescence techniques, one can also use immunohistochemistry, where a tissue section on a glass slide is incubated with an enzyme-labeled antibody.

    02:52 Binding will occur if the antibody detects antigens in the tissue section.

    03:00 The substrate for the enzyme can be added.

    03:05 The enzyme mediates a color change in this substrate.

    03:10 And this can be detected under the microscope.

    03:18 In indirect immunochemistry, the patient’s antibody is detected using an enzyme labeled anti-human immunoglobulin.

    03:28 Again the enzyme mediates a color change in the substrate which can be visualized under a microscope.

    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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