immunofluorescence - IMMUNOFLUORESCENC E Introduction...

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IMMUNOFLUORESCENC E
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Introduction Immunofluorescence is a common laboratory technique which has been ameliorated over the years by scientists such as Kaplan, Coons and Mary Osborne. This technique is used in both diagnostics in the clinic and research work.
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Introduction It can be used on both fresh as well as fixed samples. Immunofluorescence covers a wide range of uses which include: in cultured cells, suspended cells, beads as well as microarrays, which are used in detecting specific proteins.
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Introduction… In immunofluorescence, antibodies are conjugated chemically to fluorescent dyes such as tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC). The labelled antibodies are then bound, either directly or indirectly, to the antigen of specific interest.
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Introduction… This antigen-antibody complex is then detected by use of a fluorescent dye and the intensity of the florescent light is then measured through the use of a detector which also quantifies the signal such as an array scanner, automated imaging paraphernalia, confocal microscope, flow cytometer.
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TYPES OF IMMUNOFLOURESCENT TECHNIQUES There are two main techniques of immunofluorescence: o Direct immunofluorescence o Indirect immunofluorescence
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TYPES OF IMMUNOFLUORESCENT TECHNIQUES DIRECT FLOURESCENCE Used less frequently than the indirect immunofluorescent technique. The antibody used to target the molecule of interest is conjugated to the fluorescent dye.
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TYPES OF IMMUNOFLUORESCENT TECHNIQUES Upon attachment to the antibody, the conjugated dye fluoresces, leading to detection of antigen-antibody complexes. INDIRECT FLOURESCENCE In this technique, two antibodies are used: the primary antibody and the secondary antibody.
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TYPES OF IMMUNOFLUORESCENT TECHNIQUES The primary antibody, which is unconjugated, binds to the specific molecule of interest. The second antibody/anti- immunoglobulin, conjugated to the fluorescent dye, is then directed to the constant portion of the primary antibody.
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TYPES OF IMMUNOFLUORESCENCE:
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TYPES OF IMMUNOFLUORESCENCE:
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Advantages of Direct Immunofluorescence Time taken to stain the samples is shorter. The dual and triple procedures of labelling are simpler. Most preferable procedure when raising multiple antibodies in the same species, eg. , two monoclonal antibodies from mice.
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Disadvantages of Direct Immunofluorescence Lower signal generated.
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  • Summer '16
  • Felix
  • Fluorophore, Immunohistochemistry, Immunofluorescence

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