Fluorescent Immunohistochemistry

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2021 Dec 3

PMID 34859403

Citations 3

Authors

Vanessa Moreno

Elizabeth A Smith

Sergio Pina-Oviedo

Affiliations

Soon will be listed here.

Abstract

Immunofluorescence is an important immunochemical technique that utilizes fluorescence-labeled antibodies to detect specific target antigens. It is used widely in both scientific research and clinical laboratories. Immunofluorescence allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry. However, analysis of samples labeled with fluorescence-labeled antibodies has to be performed using a fluorescence microscope or other type of fluorescence imaging. There are two methods available: direct (primary) and indirect (secondary) immunofluorescence. Here, we describe the principle of immunofluorescence methods as well as the preparation of fresh-frozen and formalin-fixed, paraffin embedded tissues for both direct and indirect immunofluorescence labeling.

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References

Im K, Mareninov S, Diaz M, Yong W . An Introduction to Performing Immunofluorescence Staining. Methods Mol Biol. 2018; 1897:299-311. PMC: 6918834. DOI: 10.1007/978-1-4939-8935-5_26. View

Coons A . The beginnings of immunofluorescence. J Immunol. 1961; 87:499-503. View

Coons A, Kaplan M . Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med. 1950; 91(1):1-13. PMC: 2135948. DOI: 10.1084/jem.91.1.1. View

Burkholder P . COMPLEMENT FIXATION IN DISEASED TISSUES : I. FIXATION OF GUINEA PIG COMPLEMENT IN SECTIONS OF KIDNEY FROM HUMANS WITH MEMBRANOUS GLOMERULONEPHRITIS AND RATS INJECTED WITH ANTI-RAT KIDNEY SERUM. J Exp Med. 2009; 114(5):605-16. PMC: 2180385. DOI: 10.1084/jem.114.5.605. View

Molne J, Breimer M, Svalander C . Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies. Am J Kidney Dis. 2005; 45(4):674-83. DOI: 10.1053/j.ajkd.2004.12.019. View

Weber K, Bibring T, Osborn M . Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures. Exp Cell Res. 1975; 95(1):111-20. DOI: 10.1016/0014-4827(75)90615-1. View

GRAHAM Jr R, Karnovsky M . The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966; 14(4):291-302. DOI: 10.1177/14.4.291. View

Heyderman E . Immunoperoxidase technique in histopathology: applications, methods, and controls. J Clin Pathol. 1979; 32(10):971-8. PMC: 1145876. DOI: 10.1136/jcp.32.10.971. View

DeLellis R, STERNBERGER L, Mann R, Banks P, Nakane P . Immunoperoxidase technics in diagnostic pathology. Report of a workshop sponsored by the National Cancer Institute. Am J Clin Pathol. 1979; 71(5):483-8. DOI: 10.1093/ajcp/71.5.483. View

10.

Taylor C, Burns J . The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J Clin Pathol. 1974; 27(1):14-20. PMC: 477979. DOI: 10.1136/jcp.27.1.14. View

11.

Taylor C, Mason D . The immunohistological detection of intracellular immunoglobulin in formalin-paraffin sections from multiple myeloma and related conditions using the immunoperoxidase technique. Clin Exp Immunol. 1974; 18(3):417-29. PMC: 1537991. View

12.

Jubb A, Landon T, Burwick J, Pham T, Frantz G, Cairns B . Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization. J Pathol. 2003; 200(5):577-88. DOI: 10.1002/path.1371. View

13.

Nasr S, Galgano S, Markowitz G, Stokes M, DAgati V . Immunofluorescence on pronase-digested paraffin sections: a valuable salvage technique for renal biopsies. Kidney Int. 2006; 70(12):2148-51. DOI: 10.1038/sj.ki.5001990. View

14.

Walker P, Cavallo T, Bonsib S . Practice guidelines for the renal biopsy. Mod Pathol. 2004; 17(12):1555-63. DOI: 10.1038/modpathol.3800239. View

15.

Wu X, Liu H, Liu J, Haley K, Treadway J, Larson J . Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2002; 21(1):41-6. DOI: 10.1038/nbt764. View

16.

Vorobjev I, Buchholz K, Prabhat P, Ketman K, Egan E, Marti M . Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters. Malar J. 2012; 11:312. PMC: 3544587. DOI: 10.1186/1475-2875-11-312. View

17.

Mehes G, Lorch T, Ambros P . Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis. Cytometry. 2001; 42(6):357-62. DOI: 10.1002/1097-0320(20001215)42:6<357::aid-cyto1002>3.0.co;2-6. View

18.

Robertson D, Savage K, Reis-Filho J, Isacke C . Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol. 2008; 9:13. PMC: 2288605. DOI: 10.1186/1471-2121-9-13. View

19.

Mansfield J . Multispectral imaging: a review of its technical aspects and applications in anatomic pathology. Vet Pathol. 2013; 51(1):185-210. DOI: 10.1177/0300985813506918. View

20.

Odell I, Cook D . Immunofluorescence techniques. J Invest Dermatol. 2013; 133(1):e4. DOI: 10.1038/jid.2012.455. View