Immunofluorescence (IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens.  The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.
By conjugating the antibody to a fluorophore, the position of the target biomolecule is visualized by exciting the fluorophore and measuring the emission of light in a specific predefined wavelength using a fluorescence microscope. It is imperative that the binding of the fluorophore to the antibody itself, do not interfere with the immunological specificity of the antibody or the binding capacity of its antigen.
Immunofluorescence is a widely used example of immunostaining (using antibodies to stain proteins) and is a specific example of immunohistochemistry (the use of the antibody-antigen relationship in tissues). This technique primarily utilizes fluorophores to visualize the location of the antibodies, while others provoke a color change in the environment containing the antigen of interest or make use of a radioactive label. Immunofluorescent techniques that utilized labelled antibodies was conceptualized in the 1940’s by Albert H. Coons.
Immunofluorescence is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, cultured cell lines, or individual cells. Its usage includes analysis of the distribution of proteins, glycans, small biological and non-biological molecules, and visualization of structures such as intermediate-sized filaments.
If the topology of a cell membrane is undetermined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures within the cell membrane. Immunofluorescence (IF) can also be used as a “semi-quantitative” method to gain insight into the levels and localization patterns of DNA methylation. IF can additionally be used in combination with other, non-antibody methods of fluorescent staining, e.g., the use of DAPI to label DNA.
Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including the epifluorescence microscope, confocal microscope, and widefield microscope.