Jump to content

Immunofluorescence

From Wikipedia, the free encyclopedia
Vasculature of porcineskinunder fluorescence (Smooth muscleactinwith AlexaFluor 488). Green = smooth muscle actin (SMA) with Alexa 488 fluorophore. Blue = DAPI counterstain. Red = auto-fluorescence.

Immunofluorescence(IF)is alight microscopy-based technique that allows detection and localization of a wide variety of targetbiomoleculeswithin a cell or tissue at a quantitative level. The technique utilizes the binding specificity ofantibodiesandantigens.[1]The specific region an antibody recognizes on an antigen is called anepitope.Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.[2][3]

By conjugating the antibody to afluorophore,the position of the target biomolecule is visualized by exciting the fluorophore and measuring the emission of light in a specific predefined wavelength using afluorescence microscope.It is imperative that the binding of thefluorophoreto the antibody itself, do not interfere with the immunological specificity of the antibody or the binding capacity of its antigen.[4][5]

Immunofluorescence is a widely used example ofimmunostaining(using antibodies to stain proteins) and is a specific example ofimmunohistochemistry(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. Immunofluorescent techniques that utilized labelled antibodies was conceptualized in the 1940s byAlbert H. Coons.[2][6][7]

Photomicrograph of ahistological sectionof human skin prepared fordirect immunofluorescenceusing an anti-IgG antibody. The skin is from a patient with systemiclupus erythematosusand shows IgG deposit at two different places: The first is a band-like deposit along the epidermalbasement membrane( "lupus band test" is positive). The second is within the nuclei of theepidermalcells (anti-nuclear antibodies).

Immunofluorescence is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, culturedcell lines,or individual cells. Its usage includes analysis of the distribution ofproteins,glycans,small biological and non-biological molecules, and visualization of structures such as intermediate-sized filaments.[8]

If the topology of a cell membrane is undetermined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures within the cell membrane.[9]Immunofluorescence (IF) can also be used as a “semi-quantitative” method to gain insight into the levels and localization patterns of DNA methylation. IF can additionally be used in combination with other, non-antibody methods of fluorescent staining, e.g., the use ofDAPIto labelDNA.[10][11]

Examination of immunofluorescence specimens can be conducted utilizing various microscope configurations, including theepifluorescence microscope,confocal microscope,and widefield microscope.[12]

Types

[edit]
Mainantinuclear antibodypatterns on immunofluorescence.[13]

Preparation of fluorescence

[edit]

To perform immunofluorescence staining, afluorophoremust 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).[1][2]The following descriptions will focus primarily on these classes in terms of conjugated antibodies.[12]

Primary (direct)

[edit]
Basic concept ofPrimary Immunofluorescence:An antibody with a conjugated fluorophore, that is specifically bound to an epitope on the target molecule.

Primary (direct) immunofluorescence (DIF) uses a single antibody, conjugated to afluorophore.The antibody recognizes the target molecule (antigen) and binds to a specific region, called theepitope.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.[1][14]

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.[12]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[2][12]when compared to Secondary (Indirect) Immunofluorescence.[1]

Basic concept ofSecondary Immunofluorescence:Secondary antibody, with a conjugated fluorophore, bound to a primary antibody that is specifically bound to an epitope on the target molecule.

Secondary (indirect)

[edit]

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.[1]

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.[1]

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.[1]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).[15][16]

Limitations

[edit]
Basic concept of theABC-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.

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.[17]To study structures within living cells, in combination with fluorescence, one can utilizerecombinant proteinscontaining fluorescent protein domains, e.g.,green fluorescent protein(GFP). The GFP-technique involves altering the genetic information of the cells.[18][19]

A significant problem with immunofluorescence isphotobleaching,[12]the fluorophores permanent loss of ability to emit light.[1]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 asAlexa Fluors,Seta Fluors, orDyLight Fluors.[2]

Basic concept of theLSAB-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.

Other problems that may arise when using immunofluorescence techniques includeautofluorescence,spectral overlap and non-specific staining.[1][2]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.[2][4][1]

Advances

[edit]

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.[20]

Photomicrograph of ahistological sectionof human skin prepared fordirect immunofluorescenceusing an anti-IgA antibody. The skin is from a patient withHenoch–Schönlein purpura:IgA deposits are found in the walls of small superficial capillaries (yellow arrows). The pale wavy green area on top is theepidermis,the bottom fibrous area is thedermis.

Super-resolution fluorescence microscopymethods can produce images with a higher resolution than those microscopes imposed by thediffraction limit.This enables the determination of structural details within the cell.[21]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).[22]

Notable people

[edit]

See also

[edit]

References

[edit]
  1. ^abcdefghijOdell ID, Cook D (2013-01-01)."Immunofluorescence Techniques".Journal of Investigative Dermatology.133(1): e4.doi:10.1038/jid.2012.455.PMID23299451.
  2. ^abcdefgJoshi S, Yu D (2017),"Immunofluorescence",Basic Science Methods for Clinical Researchers,Elsevier, pp. 135–150,doi:10.1016/b978-0-12-803077-6.00008-4,ISBN978-0-12-803077-6,retrieved2024-02-14
  3. ^Ladner RC (2007-01-01). "Mapping the epitopes of antibodies".Biotechnology & Genetic Engineering Reviews.24(1): 1–30.CiteSeerX10.1.1.536.6172.doi:10.1080/02648725.2007.10648092.PMID18059626.S2CID34595289.
  4. ^abMarks KM, Nolan GP (August 2006)."Chemical labeling strategies for cell biology".Nature Methods.3(8): 591–596.doi:10.1038/nmeth906.ISSN1548-7091.PMID16862131.S2CID27848267.
  5. ^Owenius R, Österlund M, Lindgren M, Svensson M, Olsen OH, Persson E, Freskgård PO, Carlsson U (October 1999)."Properties of Spin and Fluorescent Labels at a Receptor-Ligand Interface".Biophysical Journal.77(4): 2237–2250.Bibcode:1999BpJ....77.2237O.doi:10.1016/S0006-3495(99)77064-5.PMC1300504.PMID10512843.
  6. ^Hökfelt T (November 1999)."Neurobiology thanks to microbiology: The legacy of Albert H. Coons (1912–1978)".Brain Research Bulletin.50(5–6): 371–372.doi:10.1016/S0361-9230(99)00109-4.PMID10643440.S2CID33618171.
  7. ^Sheng W, Zhang C, Mohiuddin TM, Al-Rawe M, Zeppernick F, Falcone FH, Meinhold-Heerlein I, Hussain AF (2023-02-04)."Multiplex Immunofluorescence: A Powerful Tool in Cancer Immunotherapy".International Journal of Molecular Sciences.24(4): 3086.doi:10.3390/ijms24043086.ISSN1422-0067.PMC9959383.PMID36834500.
  8. ^Franke WW, Schmid E, Osborn M, Weber K (October 1978)."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.Bibcode:1978PNAS...75.5034F.doi:10.1073/pnas.75.10.5034.PMC336257.PMID368806.
  9. ^Wang H, Lee EW, Cai X, Ni Z, Zhou L, Mao Q (December 2008)."Membrane topology of the human breast cancer resistance protein (BCRP/ABCG2) determined by epitope insertion and immunofluorescence".Biochemistry.47(52): 13778–13787.doi:10.1021/bi801644v.PMC2649121.PMID19063604.
  10. ^Çelik S (January 2015)."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:10.1016/j.jim.2014.11.011.PMID25435341.
  11. ^Grimason A, Smith H, Parker J, Bukhari Z, Campbell A, Robertson L (March 1994)."Application of DAPI and immunofluorescence for enhanced identification of Cryptosporidium spp oocysts in water samples".Water Research.28(3): 733–736.Bibcode:1994WatRe..28..733G.doi:10.1016/0043-1354(94)90154-6.
  12. ^abcdePiña R, Santos-Díaz AI, Orta-Salazar E, Aguilar-Vazquez AR, Mantellero CA, Acosta-Galeana I, Estrada-Mondragon A, Prior-Gonzalez M, Martinez-Cruz JI, Rosas-Arellano A (2022-01-26)."Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence".International Journal of Molecular Sciences.23(3): 1426.doi:10.3390/ijms23031426.ISSN1422-0067.PMC8836139.PMID35163349.
  13. ^Al-Mughales JA (2022)."Anti-Nuclear Antibodies Patterns in Patients With Systemic Lupus Erythematosus and Their Correlation With Other Diagnostic Immunological Parameters".Front Immunol.13:850759.doi:10.3389/fimmu.2022.850759.PMC8964090.PMID35359932.
    Minor edits by Mikael Häggström, MD
    - Attribution 4.0 International (CC BY 4.0) license
  14. ^"Immunohistochemical Staining Methods"(PDF).IHC Guidebook(Sixth ed.). Dako Denmark A/S, An Agilent Technologies Company. 2013. Archived fromthe original(PDF)on 2016-08-03.Retrieved2014-05-14.
  15. ^Im K, Mareninov S, Diaz MF, Yong WH (2019), Yong WH (ed.), "An Introduction to Performing Immunofluorescence Staining",Biobanking,vol. 1897, New York, NY: Springer New York, pp. 299–311,doi:10.1007/978-1-4939-8935-5_26,ISBN978-1-4939-8933-1,PMC6918834,PMID30539454
  16. ^Yarilin D, Xu K, Turkekul M, Fan N, Romin Y, Fijisawa S, Barlas A, Manova-Todorova K (2015-03-31)."Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection".Scientific Reports.5(1): 9534.Bibcode:2015NatSR...5E9534Y.doi:10.1038/srep09534.ISSN2045-2322.PMC4821037.PMID25826597.
  17. ^"Fixation and Permeabilization in in[sic] Immunocytochemistry/Immunofluorescence (ICC/IF)".Novus Biologicals.2024-02-14.Retrieved2024-02-14.
  18. ^Ehrhardt D (December 2003)."GFP technology for live cell imaging".Current Opinion in Plant Biology.6(6): 622–628.Bibcode:2003COPB....6..622E.doi:10.1016/j.pbi.2003.09.014.PMID14611963.
  19. ^Chalfie M (October 1995). "Green fluorescent protein".Photochemistry and Photobiology.62(4): 651–656.doi:10.1111/j.1751-1097.1995.tb08712.x.PMID7480149.S2CID3944607.
  20. ^Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Normanno D, Singer RH, Lionnet T, Lavis LD (March 2015)."A general method to improve fluorophores for live-cell and single-molecule microscopy".Nature Methods.12(3): 244–250.doi:10.1038/nmeth.3256.ISSN1548-7091.PMC4344395.PMID25599551.
  21. ^Huang B, Bates M, Zhuang X (2009-06-02)."Super-resolution fluorescence microscopy".Annual Review of Biochemistry.78:993–1016.doi:10.1146/annurev.biochem.77.061906.092014.PMC2835776.PMID19489737.
  22. ^Leung BO, Chou KC (2011-09-01)."Review of Super-Resolution Fluorescence Microscopy for Biology".Applied Spectroscopy.65(9): 967–980.Bibcode:2011ApSpe..65..967L.doi:10.1366/11-06398.PMID21929850.S2CID5545465.
[edit]
Retrieved from "https://en.wikipedia.org/w/index.php?title=Immunofluorescence&oldid=1220066109"