9. Complement Regulators and Receptors in Tissues
| Abstract |
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Complement regulators and receptors can be identified individually in tissues by probing thin (5 μm) tissue sections fixed
on a solid glass support with appropriate antibodies. The bound antibodies are visualized with a proper label that has been
attached to the primary antibody itself or to a secondary antibody against the primary immunoglobulin (see
Note 1). Despite the label the antibody retains its reactivity with its antigen. In immunofluorescence microscopy the label is a
fluorochrome that emits visible light under exposure to exciting radiation like ultraviolet (UV) light. Washing out the extra
stain reveals the sites where antibody has contacted the antigen. These sites appear as brightly illuminated areas in contrast
to the dark background (see
Notes 2 and 3). The color of the light or fluorescence depends on the chromogen used: Fluorescein iso-thiocyanate (FITC) excites at 495
nm and emits green light (peak at 525 nm) whereas tetra-methyl rhodamine-iso-thiocyanate (TRITC) absorbs maximally at 552
nm and gives red fluorescence (peak at 570 nm). To detect fluorochrome labels special microscopes equipped with an UV-light
source and appropriate filters are required. In immunoenzymatic stainings enzymes conjugated to antibodies react with appropriate
substrates and generate color deposits on the samples. Distribution of the target antigens can thus be examined with a light
microscope (see
Note 4).
Affiliation(s): (2) Department of Bacteriology and Immunology,,Haartman Institute, University of Helsinki, Helsinki, Finland
Book Title: Complement Methods and Protocols
Series: Methods in Molecular Biology | Volume: 150 | Pub. Date: Mar-30-2000 | Page Range: 123-129 | DOI: 10.1385/1-59259-056-X:123
Subject: Immunology
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