2. 2
Immunofluorescence
• If antibody molecules are tagged with a fluorescent dye,
immune complexes containing these fluorescently labeled
antibodies (FA) can be detected by colored light emission
when excited by light of the appropriate wavelength.
• Antibody molecules bound to antigens in cells or tissue
sections can similarly be visualized.
• The emitted light can be viewed with a fluorescence
microscope, which is equipped with an UV light source.
• In this technique, known as immunofluorescence, fluorescent
compounds such as:
fluorescein
rhodamine,
and phycoerythrin,
• are coupled to Abs which then ca be detected by
fluorescence microscope
4. 4
Immunofluorescence
• Fluorescent-antibody can be used for staining of
cell membrane molecules or tissue sections
• In this way, the distribution of antigen throughout
a tissue and within cells can be demonstrated.
• The method can also be used for the detection
of antibodies directed against antigens already
known to be present in a given tissue section or
cell preparation.
• There are two types of IF:
• Direct
• Indirect
5. 5
Direct immunofluorescence
• The antibody to the tissue antigen is
conjugated with the fluorochrome and
applied directly.
• For example, to show the distribution of a
thyroid autoantigen reacting with the
autoantibodies present in the serum of a
patient with Hashimoto's disease, a type
of thyroid autoimmunity.
• Isolate IgG from the patient's serum,
conjugate it with fluorescein, and apply it
to a section of human thyroid on a slide.
• When viewed in the fluorescence
microscope, the cytoplasm of the follicular
epithelial cells will be brightly stained.
7. 7
Indirect immunofluorescence
• In this double-layer technique,
– the unlabeled antibody is applied directly to the tissue
substrate
– and visualized by treatment with a fluorochrome-
conjugated anti-immunoglobulin serum
• A number of reagents have been developed for
indirect staining.
• The most common is a fluorochrome-labeled
secondary antibody raised in one species
against antibodies of another species.
8. 8
Indirect immunofluorescence
• In this case, in order to find out whether or
not the serum of a patient has antibodies
to thyroid epithelial cells,
• First treat a thyroid section with the serum,
• wash well and then apply a fluorescein-
labeled rabbit anti-human immunoglobulin;
• If antibodies were present, there would be
staining of the thyroid epithelial cells.
9. 9
Indirect Immunofluorescence
• Ab to tissue Ag is unlabeled
• Fluorochrome-labeled anti-Ig is used to detect binding of
the first Ab.
Ag
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab
11. 11
Advantages of Indirect IF
• In the first place the fluorescence is brighter than with
the direct test since several fluorescent anti-
immunoglobulins bind on to each of the antibody
molecules present in the first layer.
• Second, even when many sera have to be screened for
specific antibodies it is only necessary to purchase a
single labeled reagent.
• The primary antibody does not need to be conjugated
with a fluorochrome. Because the supply of primary
antibody is often a limiting factor, indirect methods avoid
the loss of antibody that usually occurs during the
conjugation reaction.
• One can also test for complement fixation on the tissue
section by adding a mixture of the first antibody plus a
source of complement, followed by a fluorescent
anticomplement reagent as the second layer.
12. 12
Immunofluorescence
• Fix specimen on slide
• Add antibody specific
for the desired
antigen
• Look for fluorescence
• Fix specimen on slide
• Add antibody specific
for the desired
antigen
• Add second antibody
• Look for fluorescence
Direct Indirect
13. 13
• Immunofluorescence has been applied to
identify a number of subpopulations of
lymphocytes,
– notably the CD4+ and CD8+ T-cell subpopulations.
– The technique is also suitable for identifying bacterial
species,
– detecting Ag-Ab complexes in autoimmune disease,
– detecting complement components in tissues,
– and localizing hormones and other cellular products
stained in situ.
• A major application of the fluorescent-antibody
technique is the localization of antigens in tissue
sections or in subcellular compartments.
Applications: Immunofluorescence
