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Immmunoflorescence Assay

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  2. 2.  Immunofluorescence is an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies.  The specificity of antibodies to their antigen is the base for immunofluorescence.  The biological samples include tissue and cells.  Immunofluorescence allows researchers to evaluate whether or not cells in a particular sample express the antigen in question.  In cases where an immune positive signal is found, immunofluorescence also allows researchers to determine which subcellular compartments are expressing the antigen.  Immunofluorescence can be used on cultured cell lines, tissue sections, or individual cells.  Immunofluorescence may be used to analyse the distribution of proteins, glycans, and small biological and non-biological molecules.  Immunofluorescence has been widely used in biological research and medical research yield and becomes one most important and effective method.
  3. 3. TWO DIFFERENT IMMUNOFLUORESCENCE ASSAY Direct Immunofluorescence Assay Indirect Immunofluorescence Assay
  4. 4. DIRECT IMMUNOFLUORESCENCE  The antibody is itself conjugated with the fluorochrome and applied directly to a monolayer of cells or to frozen tissue on a slide.  When examined with a fluorescence microscope, the antibody labeled with the fluorescent compound identifies the localized antigen.
  5. 5. INDIRECT IMMUNOFLUORESCENCE  Unlike direct immunofluorescence, indirect immunofluoresence is a double-layer technique.  The unlabeled antibody is applied directly to the tissue substrate and then treated with a fluorochrome-conjugated anti-IgG.  There are several advantages to this technique, and it is typically used more frequently than the direct method.  Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, this produces brighter fluorescence than in the direct method.  It is also more time-efficient since there is only one fluorescent-labeled reagent, the anti-IgG prepared during the lengthy conjugation process.