Immunofluorescence is an assay that uses antibodies to detect antigens in cells and tissues through fluorescence microscopy. It allows researchers to determine if cells express certain antigens and the subcellular localization of antigens. Immunofluorescence can be performed on cell lines, tissue sections, or individual cells. There are two main types: direct immunofluorescence uses antibodies directly conjugated to fluorophores, while indirect immunofluorescence uses unlabeled primary antibodies detected by fluorophore-conjugated secondary antibodies, producing brighter signals. Both techniques localize antigens in biological samples through fluorescence.

1. IMMUNOFLORESCENCE

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. TWO DIFFERENT IMMUNOFLUORESCENCE ASSAY
Direct
Immunofluorescence
Assay
Indirect
Immunofluorescence
Assay

5. 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.

7. 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.