Immunofluorescence

File:Main antinuclear antibody patterns on immunofluorescence.png patterns on immunofluorescence.{{cite journal| author=Al-Mughales JA| title=Anti-Nuclear Antibodies Patterns in Patients With Systemic Lupus Erythematosus and Their Correlation With Other Diagnostic Immunological Parameters. | journal=Front Immunol | year= 2022 | volume= 13 | issue= | pages= 850759 | pmid=35359932 | doi=10.3389/fimmu.2022.850759 | pmc=8964090 | doi-access=free }}
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Mikael Häggström, MD
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To perform immunofluorescence staining, a fluorophore must be conjugated (“tagged”) to an antibody. Staining procedures can be applied to both retained intracellular expressed antibodies, or to cell surface antigens on living cells. There are two general classes of immunofluorescence techniques: primary (direct) and secondary (indirect). The following descriptions will focus primarily on these classes in terms of conjugated antibodies.

File:Primary Immunofluorescence.png

Primary (direct) immunofluorescence (DIF) uses a single antibody, conjugated to a fluorophore. The 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 type of fluorophore, will emit a specific wavelength of light once excited.{{cite book |title=IHC Guidebook |date=2013 |publisher=Dako Denmark A/S, An Agilent Technologies Company |edition=Sixth |chapter=Immunohistochemical Staining Methods |access-date=2014-05-14 |chapter-url=http://www.dako.com/08002_03aug09_ihc_guidebook_5th_edition_chapter_10.pdf |archive-url=https://web.archive.org/web/20160803131150/http://www.dako.com/08002_03aug09_ihc_guidebook_5th_edition_chapter_10.pdf |archive-date=2016-08-03 |url-status=dead}}

The direct attachment of the fluorophore to the antibody reduces the number of steps in the sample preparation procedure, saving time and reducing non-specific background signal during analysis. This also limits the possibility of antibody cross-reactivity, and possible mistakes throughout the process. One disadvantage of DIF is the limited number of antibodies that can bind to the antigen. This limitation may reduce sensitivity to the technique. When the target protein is available in only small concentrations, a better approach would be secondary IF, which is considered to be more sensitive than DIF when compared to Secondary (Indirect) Immunofluorescence.

File:Secondary Immunofluorescence.png

Secondary (indirect) immunofluorescence (SIF) is similar to direct immunofluorescence, however the technique utilizes two types of antibodies whereas only one of them have a conjugated fluorophore. The antibody with the conjugated fluorophore is referred to as the secondary antibody, while the unconjugated is referred to as the primary antibody.

The principle of this technique is that the primary antibody specifically binds to the epitope on the target molecule, whereas the secondary antibody, with the conjugated fluorophore, recognizes and binds to the primary antibody.  

This technique is considered to be more sensitive than primary immunofluorescence, because multiple secondary antibodies can bind to the same primary antibody. The increased number of fluorophore molecules per antigen increases the amount of emitted light, and thus amplifies the signal. There are different methods for attaining a higher fluorophore-antigen ratio such as the Avidin-Biotin Complex (ABC method) and Labeled Streptavidin-Biotin (LSAB method).{{Citation |last1=Im |first1=Kyuseok |title=An Introduction to Performing Immunofluorescence Staining |date=2019 |work=Biobanking |volume=1897 |pages=299–311 |editor-last=Yong |editor-first=William H. |place=New York, NY |publisher=Springer New York |doi=10.1007/978-1-4939-8935-5_26 |isbn=978-1-4939-8933-1 |pmc=6918834 |pmid=30539454 |last2=Mareninov |first2=Sergey |last3=Diaz |first3=M. Fernando Palma |last4=Yong |first4=William H.}}{{Cite journal |last1=Yarilin |first1=Dmitry |last2=Xu |first2=Ke |last3=Turkekul |first3=Mesruh |last4=Fan |first4=Ning |last5=Romin |first5=Yevgeniy |last6=Fijisawa |first6=Sho |last7=Barlas |first7=Afsar |last8=Manova-Todorova |first8=Katia |date=2015-03-31 |title=Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection |journal=Scientific Reports |language=en |volume=5 |issue=1 |page=9534 |doi=10.1038/srep09534 |issn=2045-2322 |pmc=4821037 |pmid=25826597|bibcode=2015NatSR...5E9534Y }}

File:ABC-method.png

Immunofluorescence is only limited to fixed (i.e. dead) cells, when studying structures within the cell, as antibodies generally do not penetrate intact cellular or subcellular membranes in living cells, because they are large proteins. To visualize these structures, antigenic material must be fixed firmly on its natural localization inside the cell.{{Cite web |date=2024-02-14 |title=Fixation and Permeabilization in in[sic] Immunocytochemistry/Immunofluorescence (ICC/IF) |url=https://www.novusbio.com/support/fixation-and-permeabilization-in-icc-if |access-date=2024-02-14 |website=Novus Biologicals}} To study structures within living cells, in combination with fluorescence, one can utilize recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). The GFP-technique involves altering the genetic information of the cells.{{Cite journal |last=Ehrhardt |first=David |date=December 2003 |title=GFP technology for live cell imaging |url=https://linkinghub.elsevier.com/retrieve/pii/S1369526603001171 |journal=Current Opinion in Plant Biology |language=en |volume=6 |issue=6 |pages=622–628 |doi=10.1016/j.pbi.2003.09.014|pmid=14611963 |bibcode=2003COPB....6..622E |url-access=subscription }}{{cite journal |vauthors=Chalfie M |date=October 1995 |title=Green fluorescent protein |journal=Photochemistry and Photobiology |volume=62 |issue=4 |pages=651–656 |doi=10.1111/j.1751-1097.1995.tb08712.x |pmid=7480149 |s2cid=3944607}}

A significant problem with immunofluorescence is photobleaching, the fluorophores permanent loss of ability to emit light. To mitigate the risk of photobleaching one can employ different strategies. By reducing or limiting the intensity, or timespan of light exposure, the absorption-emission cycle of fluorescent light is decreased, thus preserving the fluorophores functionality. One can also increase the concentration of fluorophores, or opt for more robust fluorophores that exhibit resilience against photobleaching such as Alexa Fluors, Seta Fluors, or DyLight Fluors.

File:LSAB-method.jpg

Other problems that may arise when using immunofluorescence techniques include autofluorescence, spectral overlap and non-specific staining. Autofluorescence includes the natural fluorescence emitted from the sample tissue or cell itself. Spectral overlap happens when a fluorophore has a broad emission specter, that overlaps with the specter of another fluorophore, thus giving rise to false signals. Non-specific staining occurs when the antibody, containing the fluorophore, binds to unintended proteins because of sufficient similarity in the epitope. This can lead to false positives.

The main improvements to immunofluorescence lie in the development of fluorophores and fluorescent microscopes. Fluorophores can be structurally modified to improve brightness and photostability, while preserving spectral properties and cell permeability.{{Cite journal |last1=Grimm |first1=Jonathan B |last2=English |first2=Brian P |last3=Chen |first3=Jiji |last4=Slaughter |first4=Joel P |last5=Zhang |first5=Zhengjian |last6=Revyakin |first6=Andrey |last7=Patel |first7=Ronak |last8=Macklin |first8=John J |last9=Normanno |first9=Davide |last10=Singer |first10=Robert H |last11=Lionnet |first11=Timothée |last12=Lavis |first12=Luke D |date=March 2015 |title=A general method to improve fluorophores for live-cell and single-molecule microscopy |journal=Nature Methods |language=en |volume=12 |issue=3 |pages=244–250 |doi=10.1038/nmeth.3256 |issn=1548-7091 |pmc=4344395 |pmid=25599551}}

File:HSP IF IgA.jpg 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.]]

Super-resolution fluorescence microscopy methods can produce images with a higher resolution than those microscopes imposed by the diffraction limit. This enables the determination of structural details within the cell.{{cite journal |vauthors=Huang B, Bates M, Zhuang X |date=2009-06-02 |title=Super-resolution fluorescence microscopy |journal=Annual Review of Biochemistry |volume=78 |pages=993–1016 |doi=10.1146/annurev.biochem.77.061906.092014 |pmc=2835776 |pmid=19489737}} Super-resolution in fluorescence, more specifically, refers to the ability of a microscope to prevent the simultaneous fluorescence of adjacent spectrally identical fluorophores (spectral overlap). Some of the 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).{{Cite journal |last1=Leung |first1=Bonnie O. |last2=Chou |first2=Keng C. |date=2011-09-01 |title=Review of Super-Resolution Fluorescence Microscopy for Biology |url=http://journals.sagepub.com/doi/10.1366/11-06398 |journal=Applied Spectroscopy |language=en |volume=65 |issue=9 |pages=967–980 |doi=10.1366/11-06398|pmid=21929850 |bibcode=2011ApSpe..65..967L |s2cid=5545465 |url-access=subscription }}

• Albert Hewett Coons (1912–1978), physician, pathologist and immunologist
• Cornelia Mitchell Downs (1892–1987), microbiologist and journalist

• Antibodies
• Cutaneous conditions with immunofluorescence findings
• Fluorescence
• Immunochemistry
• Immunohistochemistry
• Patching and Capping

{{Reflist}}

• [http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/ Images associated with autoimmune diseases] {{Webarchive|url=https://web.archive.org/web/20090925190032/http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/ |date=2009-09-25 }} at University of Birmingham
• [http://www.bio.davidson.edu/COURSES/genomics/method/IMF.html Overview] at Davidson College
• {{MeshName|Immunofluorescence}}

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