Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. Histology (from the Greek = 'tissue' is the study of the microscopic anatomy of cells and tissues of Plants and The skin is from a patient with Henoch-Schonlein purpura : IgA deposits are found in the walls of small superficial capillaries (yellow arrows). Henoch-Schönlein purpura, also known as allergic purpura and commonly abbreviated to HSP, is a systemic Vasculitis (inflammation of blood vessels characterized The pale wavy green area on top is the epidermis, the bottom fibrous area is the dermis. The dermis is a layer of Skin beneath the epidermis that consists of Connective tissue, and cushions the body from stress and strain

Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgG antibody. Histology (from the Greek = 'tissue' is the study of the microscopic anatomy of cells and tissues of Plants and 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). Systemic lupus erythematosus ( SLE or lupus,) is a chronic autoimmune disease that can be fatal though with recent medical advances fatalities are becoming The basement membrane is a structure that supports overlying Epithelial or Endothelial cells.

Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily An antigen (from antibody-generating) or immunogen is a substance that prompts the generation of Antibodies and can cause an immune response Fluorescence is a Luminescence that is mostly found as an A dye can generally be described as a Colored substance that has an affinity to the substrate to which it is being applied This technique is often used to visualize the subcellular distribution of biomolecules of interest. A biomolecule is any organic Molecule that is produced by living Organisms including large Polymeric molecules such as Proteins Immunofluorescent-labelled tissue sections or cultures are studied using a fluorescence microscope or by confocal microscopy. A fluorescence microscope (colloquially synonymous with epifluorescent microscope) is a light Microscope used to study properties of organic or inorganic substances Confocal microscopy is an optical imaging technique used to increase Micrograph contrast and/or to Reconstruct three-dimensional Images by

Most commonly, immunofluorescence employs two sets of antibodies: a primary antibody is used against the antigen of interest; a subsequent, secondary, dye-coupled antibody is introduced that recognizes the primary antibody. In this fashion the researcher may create several primary antibodies that recognize various antigens, but, because they all share a common constant region, may be recognized by a single dye-coupled antibody. Typically this is done by using antibodies made in different species. For example, a researcher might create antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit antibodies that recognize the goat antibody constant region (denoted rabbit anti-goat). This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.

In some cases, it is advantageous to use primary antibodies directly labelled with a fluorophore. A fluorophore, in analogy to a Chromophore, is a component of a molecule which causes a molecule to be Fluorescent. This direct labelling decreases the number of steps in the staining procedure and, more importantly, often avoids cross-reactivity and high background problems. Fluorescent labelling can be performed in less than one hour with readily available labeling kits. Fluorescent labelling is the process of covalently attaching a Fluorophore to another molecule such as a Protein or Nucleic acid.

As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Photobleaching is the photochemical destruction of a Fluorophore. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e. g. Alexa Fluors or DyLight Fluors). History The Alexa Fluor dyes were named after Alex Haugland son of the founders of Molecular Probes Richard and Rosaria Haugland

Many uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein domains, e. Recombinant DNA is a form of synthetic DNA that is engineered through the combination or insertion of one or more DNA strands thereby combining DNA sequences g. green fluorescent protein (GFP). The green fluorescent protein ( GFP) is composed of 238 Amino acids (26 Use of such "tagged" proteins allows much better localization and less disruption of protein function.

Protocol

Immunostaining protocol

External links

• Images associated with autoimmune diseases at University of Birmingham
• Immunofluorescence protocol and confocal microscopy resources at confocal-microscopy. The University of Birmingham (informally Birmingham University) is a British red brick University located in the city of Birmingham org
• Overview at Davidson College
• Immunofluorescence Staining Protocol

• MeSH Immunofluorescence