SOLUTIONS TO EXERCISES, LESSON 6
Immunofluorescence is a method of detecting an antigen or antibody in tissue by
the pattern of fluorescence resulting when the tissue is exposed to the specific
antibody or antigen-labeled with a fluorochrome such as fluorescein.
Immunofluorescence can be qualitative or quantitative. In a qualitative procedure,
a fluorescent microscope is necessary to visualize the presence of a labeled
antigen or antibody in the specimen. Fluorescence is the emission of light of one
color from a substance being exposed to light of a different color or wavelength.
One of the most common fluorochromes is fluorescein isothiocyanate.
The two most common methods used in the performance of immunofluorescent
microscopy are the direct and indirect techniques.
In the direct method, the antibody is labeled with a fluorescent compound and is
used to detect the presence of antigen in tissue fixed to a slide. The direct
technique utilizes biopsy material obtained from a patient. The fluorescent-labeled
antibodies are added to the antigen in an optimal dilution and allowed to react.
The preparation is washed to remove any unreacted labeled antibodies. The
tissue sections are blotted and the preparation mounted with buffered glycerol for
examination with the fluorescent microscope.
The indirect method is used for the detection of serum antibodies utilizing an
antigen-containing substrate and a fluorescein-labeled antibody specific for human
immunoglobulins. The specific antigen-antibody (unlabeled) reaction may be
visualized by the addition of labeled antihuman globulin directed against the
antibody in the primary reaction. The antigen substrate plus patient's serum
antibody plus labeled antihuman immunoglobulin complex results in fluorescence
and detection of the specific patient's antibody in question.
Two types of microscopes used in immunofluorescent techniques are the
transmitted light microscope and the incident light or epi-illumination microscope.
One major difference is the direction from which the light or energy strikes the
specimen. In the transmitted light microscope, light strikes the specimen from
below through a condenser. In the incident light microscope, light strikes the
specimen from above, passing through the objective; this eliminates the need for a
condenser and also eliminates the problems of centering a condenser. The
incident microscope has more brightness, clearer images, and greater
fluorescence since illumination and observation of the specimen are made from
the same direction.