Microphotograph of a histological section of human skin prepared for direct immunofluorescence using an anti-IgA antibody. The skin is from a patient with Henoch-Schonlein 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.

Microphotograph 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). Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. This technique is often used to visualize subcellular distribution of biomolecules of interest. Immunofluorescent-labelled tissue sections are studied using a fluorescence microscope or by confocal microscopy. 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. 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. As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. 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). Many uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein domains, e.g. green fluorescent protein (GFP). Use of such "tagged" proteins allows much better localization and less disruption of protein function. [edit] External links Images associated with autoimmune diseases at University of Birmingham Immunofluorescence protocol and confocal microscopy resources atb confocal-microscopy.org Overview at Davidson College Immunofluorescence Staining Protocol MeSH Immunofluorescence v ? d ? e Pathology Principles of pathology Disease - Necrosis - Infection - Ischemia - Inflammation - Wound healing - Neoplasia Anatomical pathology Surgical pathology - Cytopathology - Autopsy - Molecular pathology - Forensic pathology - Dental pathology  Gross examination - Histopathology - Immunohistochemistry - Electron microscopy - Immunofluorescence - Fluorescent in situ hybridization Clinical pathology Clinical chemistry - Hematopathology - Transfusion medicine - Medical microbiology - Diagnostic immunology Enzyme assay - Mass spectrometry - Chromatography - Flow cytometry - Blood bank - Microbiological culture - Serology v ? d ? e Immunologic techniques and tests - diagnostic immunology Immunoprecipitation Chromatin immunoprecipitation - Immunodiffusion (Ouchterlony double immuno diffusion, Radial immunodiffusion, Immunoelectrophoresis, Counterimmunoelectrophoresis) Immunoassay ELISA - Enzyme Multiplied Immunoassay Technique - RAST test - Radioimmunoassay - Immunofluorescence Other Nephelometry - Agglutination (Hemagglutination) - Complement fixation test - Immunohistochemistry