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Immunofluorescence

Vasculature of porcine skin under fluorescence (Smooth muscle actin with AlexaFluor 488). Green = smooth muscle actin (SMA) with Alexa 488 fluorophore. Blue = DAPI counterstain. Red = auto-fluorescence.

Immunofluorescence (IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens.[1] The specific region an antibody recognizes on an antigen is called an epitope. 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 a fluorophore, the position of the target biomolecule is visualized by exciting the fluorophore and measuring the emission of light in a specific predefined wavelength using a fluorescence microscope. It is imperative that the binding of the fluorophore to the antibody itself does not interfere with the immunological specificity of the antibody or the binding capacity of its antigen.[4][5]

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 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 by Albert H. Coons.[2][6][7]

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).

Immunofluorescence is employed in foundational scientific investigations and clinical diagnostic endeavors, showcasing its multifaceted utility across diverse substrates, including tissue sections, cultured cell lines, or individual cells. Its usage includes analysis of the distribution of proteins, 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 of DAPI to label DNA.[10][11]

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

  1. ^ Odell ID, Cook D (2013-01-01). "Immunofluorescence Techniques". Journal of Investigative Dermatology. 133 (1): e4. doi:10.1038/jid.2012.455. PMID 23299451.
  2. ^ a b Joshi 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, ISBN 978-0-12-803077-6, retrieved 2024-02-14
  3. ^ Ladner RC (2007-01-01). "Mapping the epitopes of antibodies". Biotechnology & Genetic Engineering Reviews. 24 (1): 1–30. CiteSeerX 10.1.1.536.6172. doi:10.1080/02648725.2007.10648092. PMID 18059626. S2CID 34595289.
  4. ^ Marks KM, Nolan GP (August 2006). "Chemical labeling strategies for cell biology". Nature Methods. 3 (8): 591–596. doi:10.1038/nmeth906. ISSN 1548-7091. PMID 16862131. S2CID 27848267.
  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. PMC 1300504. PMID 10512843.
  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. PMID 10643440. S2CID 33618171.
  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. ISSN 1422-0067. PMC 9959383. PMID 36834500.
  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. PMC 336257. PMID 368806.
  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. PMC 2649121. PMID 19063604.
  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. PMID 25435341.
  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. ^ Piña 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. ISSN 1422-0067. PMC 8836139. PMID 35163349.

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تألق مناعي Arabic Imunoflurescencija BS Immunofluorescència Catalan Imunofluorescence Czech Immunfluoreszenz German Inmunofluorescencia Spanish Immunofluorestsents ET Immunofluoreszentzia EU Immunofluorescence French Inmunofluorescencia GL

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