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immunofluorescence, immunofluorescence protocol
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample The specific region an antibody recognizes on an antigen is called an epitope There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary Additionally, the binding of the fluorophore to the antibody itself cannot 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 makes use of fluorophores to visualise the location of the antibodies

Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, and may be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules This technique can even be used to visualize structures such as intermediate-sized filaments If the topology of a cell membrane has yet to be determined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures Immunofluorescence can also be used as a "semi-quantitative" method to gain insight into the levels and localization patterns of DNA methylation since it is a more time consuming method than true quantitative methods and there is some subjectivity in the analysis of the levels of methylation Immunofluorescence can be used in combination with other, non-antibody methods of fluorescent staining, for example, use of DAPI to label DNA Several microscope designs can be used for analysis of immunofluorescence samples; the simplest is the epifluorescence microscope, and the confocal microscope is also widely used Various super-resolution microscope designs that are capable of much higher resolution can also be used


  • 1 Types
    • 11 Preparation of fluorescence
    • 12 Primary direct
    • 13 Secondary indirect
  • 2 Limitations
  • 3 Advances
  • 4 See also
  • 5 References
  • 6 External links


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

Preparation of fluorescence

To make fluorochrome-labeled antibodies, a fluorochrome must be conjugated "tagged" to the antibody Likewise, an antigen can also be conjugated to the antibody with a fluorescent probe in a technique called fluorescent antigen technique Staining procedures can apply to both fixed antigen in the cytoplasm or to cell surface antigens on live cells, called "membrane immunofluorescence" It is also possible to label the complement of the antibody-antigen complex with a fluorescent probe In addition to the element to which fluorescence probes are attached, there are two general classes of immunofluorescence techniques:primary and secondary The following descriptions will focus primarily on these classes in terms of conjugated antibodies

There are two classes of immunofluorescence techniques, primary or direct and secondary or indirect

Primary direct

Primary direct immunofluorescence uses a single, primary antibody, chemically linked to a fluorophore The primary antibody recognizes the target molecule antigen and binds to a specific region called the epitope The attached fluorophore can be detected via fluorescent microscopy, which, depending on the messenger used, will emit a specific wavelength of light once excited Direct immunofluorescence, although somewhat less common, has notable advantages over the secondary indirect procedure The direct attachment of the messenger to the antibody reduces the number of steps in the procedure, saving time and reducing non-specific background signal This also limits the possibility of antibody cross-reactivity and possible mistakes throughout the process

However, some disadvantages do exist in this method Since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is substantially less sensitive than indirect immunofluorescence and may result in false negatives Direct immunofluorescence also requires the use of much more primary antibody, which is extremely expensive, sometimes running up to $40000/mL

Secondary indirect

A fluorescent stain for actin in the smooth muscle of the skin

Secondary indirect immunofluorescence uses two antibodies; the unlabeled first primary antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognizes the primary antibody and binds to it Multiple secondary antibodies can bind a single primary antibody This provides signal amplification by increasing the number of fluorophore molecules per antigen This protocol is more complex and time-consuming than the primary or direct protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody

This protocol is possible because an antibody consists of two parts, a variable region which recognizes the antigen and constant region which makes up the structure of the antibody molecule It is important to realize that this division is artificial and in reality the antibody molecule is four polypeptide chains:two heavy chains and two light chains A researcher can generate several primary antibodies that recognize various antigens have different variable regions, but all share the same constant region All these antibodies may therefore be recognized by a single secondary antibody This saves the cost of modifying the primary antibodies to directly carry a fluorophore

Different primary antibodies with different constant regions are typically generated by raising the antibody in different species For example, a researcher might create primary antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit secondary antibodies that recognize the goat antibody constant region "rabbit anti-goat" antibodies The researcher may then create a second set of primary antibodies in a mouse that could be recognized by a separate "donkey anti-mouse" secondary antibody This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments


As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching Loss of activity caused by photobleaching can be controlled by reducing or limiting 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 eg, Alexa Fluors, Seta Fluors, or DyLight Fluors Some problems that may arise from this technique include autofluorescence, extraneous undesired specific fluorescence, and nonspecific fluorescence Autofluorescence includes fluorescence emitted from the sample tissue or cell itself Extraneous undesired specific fluorescence occurs when a targeted antigen is impure and contains antigenic contaminants Nonspecific fluorescence involves the loss of a probe's specificity due to fluorophore, from improper fixation, or from a dried out specimen

Immunofluorescence is only limited to fixed ie, dead cells when structures within the cell are to be visualized because antibodies do not penetrate the cell membrane when reacting with fluorescent labels Antigenic material must be fixed firmly on the site of its natural localization inside the cell Intact antibodies can also be too large to dye cancer cells in vivo Their size results in slow tumor penetration and long circulating half-life Research has been done investigating the use of diabodies to get around this limitation Proteins in the supernatant or on the outside of the cell membrane can be bound by the antibodies; this allows for living cells to be stained Depending on the fixative that is being used, proteins of interest might become cross-linked and this could result in either false positive or false negative signals due to non-specific binding

An alternative approach is using recombinant proteins containing fluorescent protein domains, eg, green fluorescent protein GFP Use of such "tagged" proteins allows determination of their localization in live cells Even though this seems to be an elegant alternative to immunofluorescence, the cells have to be transfected or transduced with the GFP-tag, and as a consequence they become at least S1 or above organisms that require stricter security standards in a laboratory This technique involves altering the genetic information of cells


Many improvements to this method lie in the improvement of fluorescent microscopes and fluorophores Super-resolution methods generally refer to a microscope's ability to produce resolution below the Abbe limit a limit placed on light due to its wavelength This diffraction limit is about 200-300 nm in the lateral direction and 500-700 nm in the axial direction This limit is comparable or larger than some structures in the cell, and consequently, this limit prevented scientists from determining details in their structure Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores This process effectively sharpens the point-spread function of the microscope Examples of recently developed super-resolution fluorescent microscope methods include stimulated emission depletion STED microscopy, saturated structured-illumination microscopy SSIM, fluorescence photoactivation localization microscopy FPALM, and stochastic optical reconstruction microscopy STORM

See also

  • Cutaneous conditions with immunofluorescence findings
  • Immunochemistry
  • Patching and Capping


  1. ^ Mandrell, R E; Griffiss, J M; Macher, B A 1988-07-01 "Lipooligosaccharides LOS of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes" Journal of Experimental Medicine 168 1:107–126 doi:101084/jem1681107 ISSN 0022-1007 PMC 2188965  PMID 2456365 
  2. ^ Ladner, Robert C 2007-01-01 "Mapping the Epitopes of Antibodies" Biotechnology and Genetic Engineering Reviews 24 1:1–30 doi:101080/02648725200710648092 ISSN 0264-8725 
  3. ^ a b c d Akiyoshi, Kawamura, 1983-01-01 Immunofluorescence in medical science :with 28 tab Springer ua ISBN 3540124837 OCLC 643714056 
  4. ^ "Immunofluorescence" Protocol Online 
  5. ^ Franke, W W; Schmid, E; Osborn, M; Weber, K 1978-10-01 "Different intermediate-sized filaments distinguished by immunofluorescence microscopy" Proceedings of the National Academy of Sciences of the United States of America 75 10:5034–5038 doi:101073/pnas75105034 ISSN 0027-8424 PMC 336257  PMID 368806 
  6. ^ Wang, Honggang; Lee, Eun-Woo; Cai, Xiaokun; Ni, Zhanglin; Zhou, Lin; Mao, Qingcheng 2008-12-30 "Membrane Topology of the Human Breast Cancer Resistance Protein BCRP/ABCG2 Determined by Epitope Insertion and Immunofluorescence" Biochemistry 47 52:13778–13787 doi:101021/bi801644v ISSN 0006-2960 PMC 2649121  PMID 19063604 
  7. ^ Çelik, Selcen 2015-01-01 "Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge" Journal of Immunological Methods 416:1–16 doi:101016/jjim201411011 
  8. ^ "Immunofluorescence Method" Davidson College 
  9. ^ "Immunohistochemical Staining Methods" IHC Guidebook PDF Sixth ed Dako Denmark A/S, An Agilent Technologies Company 2013 
  10. ^ a b c Fritschy J, Härtig W 2001 "Immunofluorescence" ELS doi:101038/npgels0001174 ISBN 978-0-470-01590-2 
  11. ^ a b Sonn GA, Behesnilian AS, Jiang ZK, Zettlitz KA, Lepin EJ, Bentolila LA, Knowles SM, Lawrence D, Wu AM, Reiter RE 2016 "Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen PSCA Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real Time" Clinical Cancer Research 22 6:1403–12 doi:101158/1078-0432CCR-15-0503 PMC 4794340  PMID 26490315 
  12. ^ Chalfie, Martin 1995-10-01 "Green Fluorescent Protein" Photochemistry and Photobiology 62 4:651–656 doi:101111/j1751-10971995tb08712x ISSN 1751-1097 
  13. ^ a b Huang, Bo; Bates, Mark; Zhuang, Xiaowei 2009-06-02 "Super-Resolution Fluorescence Microscopy" https://dxdoiorg/101146/annurevbiochem77061906092014 doi:101146/annurevbiochem77061906092014 PMC 2835776  PMID 19489737 Retrieved 2017-03-24  External link in |website= help
  14. ^ 1959-, Diaspro, Alberto,; van,, Zandvoort, Marc A M J Super-resolution imaging in biomedicine ISBN 9781482244359 OCLC 960719686 
  15. ^ Leung, Bonnie O; Chou, Keng C 2011-09-01 "Review of Super-Resolution Fluorescence Microscopy for Biology" Applied Spectroscopy 65 9:967–980 doi:101366/11-06398 ISSN 0003-7028 

External links

  • Images associated with autoimmune diseases at University of Birmingham
  • Immunofluorescence Staining Protocol
  • Overview at Davidson College
  • Immunofluorescence at the US National Library of Medicine Medical Subject Headings MeSH
  • SynD - Automatic synapse and neurite detection in immuno-fluorescence images

immunofluorescence, immunofluorescence and immunohistochemistry, immunofluorescence assay, immunofluorescence indirecte, immunofluorescence microscope, immunofluorescence patterns, immunofluorescence ppt, immunofluorescence procedure, immunofluorescence protocol, immunofluorescence staining

Immunofluorescence Information about


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    Immunofluorescence beatiful post thanks!


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