Immunofluorescence

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.

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.

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.

[9] Immunofluorescence (IF) can also be used as a “semi-quantitative” method to gain insight into the levels and localization patterns of DNA methylation.

To visualize these structures, antigenic material must be fixed firmly on its natural localization inside the cell.

[18][19] A significant problem with immunofluorescence is photobleaching,[12] the fluorophores permanent loss of ability to emit light.

[2] Other problems that may arise when using immunofluorescence techniques include autofluorescence, spectral overlap and non-specific staining.

Non-specific staining occurs when the antibody, containing the fluorophore, binds to unintended proteins because of sufficient similarity in the epitope.

Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability.

[20] Super-resolution fluorescence microscopy methods can produce images with a higher resolution than those microscopes imposed by the diffraction limit.

Vasculature of porcine skin under fluorescence ( Smooth muscle actin with AlexaFluor 488). Green = smooth muscle actin (SMA) with Alexa 488 fluorophore. Blue = DAPI counterstain. Red = auto-fluorescence.
Photomicrograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places: The first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive). The second is within the nuclei of the epidermal cells (anti-nuclear antibodies).
Main antinuclear antibody patterns on immunofluorescence. [ 13 ]
Basic concept of Primary Immunofluorescence : An antibody with a conjugated fluorophore, that is specifically bound to an epitope on the target molecule.
Basic concept of Secondary Immunofluorescence : Secondary antibody, with a conjugated fluorophore, bound to a primary antibody that is specifically bound to an epitope on the target molecule.
Basic concept of the ABC-method : Primary antibody binds to the antigen, before binding to the biotinylated secondary antibody. Avidin-Biotin enzyme complex (ABC) then attaches to the secondary antibody.
Basic concept of the LSAB-method : Utilizes a Streptavidin–enzyme conjugate for the identification of the biotinylated secondary antibody which is bound to the primary antibody. This approach is applicable when the Avidin–Biotin complex in the ABC method becomes too large.
Photomicrograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch–Schönlein purpura : IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is the epidermis , the bottom fibrous area is the dermis .