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Fluorescent Immunohistochemistry

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Immunohistochemistry and Immunocytochemistry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2422))

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

  1. Im K, Mareninov S, Diaz MFP, Yong WH (2019) An introduction to performing immunofluorescence staining. Methods Mol Biol 1897:299–311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Coons AH, Creech HJ, Jones RN, Berliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 45:159–170

    CAS  Google Scholar 

  3. Coons AH (1961) The beginnings of immunofluorescence. J Immunol 87:499–503

    CAS  PubMed  Google Scholar 

  4. Coons AH, Kaplan MH (1950) Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 91:1–13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Burkholder PM (1961) 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 114:605–616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Molne J, Breimer ME, Svalander CT (2005) Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies. Am J Kidney Dis 45:674–683

    Article  PubMed  Google Scholar 

  7. Weber K, Bibring T, Osborn M (1975) Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence. Cytoplasmic microtubules, vinblastine-induced paracrystals, and mitotic figures. Exp Cell Res 95:111–120

    Article  CAS  PubMed  Google Scholar 

  8. Graham RC Jr, Karnovsky MJ (1966) 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 14:291–302

    Article  CAS  PubMed  Google Scholar 

  9. Heyderman E (1979) Immunoperoxidase technique in histopathology: applications, methods, and controls. J Clin Pathol 32:971–978

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. DeLellis RA, Sternberger LA, Mann RB, Banks PM, Nakane PK (1979) Immunoperoxidase technics in diagnostic pathology. Report of a workshop sponsored by the National Cancer Institute. Am J Clin Pathol 71:483–488

    Article  CAS  PubMed  Google Scholar 

  11. Taylor CR, Burns J (1974) The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J Clin Pathol 27:14–20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Taylor CR, Mason DY (1974) The immunohistological detection of intracellular immunoglobulin in formalin-paraffin sections from multiple myeloma and related conditions using the immunoperoxidase technique. Clin Exp Immunol 18:417–429

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Jubb AM, Landon TH, Burwick J et al (2003) Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization. J Pathol 200:577–588

    Article  CAS  PubMed  Google Scholar 

  14. Nasr SH, Galgano SJ, Markowitz GS, Stokes MB, D'Agati VD (2006) Immunofluorescence on pronase-digested paraffin sections: a valuable salvage technique for renal biopsies. Kidney Int 70:2148–2151

    Article  CAS  PubMed  Google Scholar 

  15. Walker PD, Cavallo T, Bonsib SM, Ad Hoc Committee on Renal Biopsy Guidelines of the Renal Pathology S (2004) Practice guidelines for the renal biopsy. Mod Pathol 17:1555–1563

    Article  PubMed  Google Scholar 

  16. Wu X, Liu H, Liu J et al (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol 21:41–46

    Article  CAS  PubMed  Google Scholar 

  17. Osborn M (1998) Immunofluorescence microscopy of cultured cells. In: Press A (ed) Cell biology: a laboratory handbook, 2nd edn. Academic Press, New York, London, pp 462–468

    Google Scholar 

  18. Vorobjev IA, Buchholz K, Prabhat P et al (2012) Optimization of flow cytometric detection and cell sorting of transgenic plasmodium parasites using interchangeable optical filters. Malar J 11:312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mehes G, Lorch T, Ambros PF (2000) Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis. Cytometry 42:357–362

    Article  CAS  PubMed  Google Scholar 

  20. Robertson D, Savage K, Reis-Filho JS, Isacke CM (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol 9:13

    Article  PubMed  PubMed Central  Google Scholar 

  21. Mansfield JR (2014) Multispectral imaging: a review of its technical aspects and applications in anatomic pathology. Vet Pathol 51:185–210

    Article  CAS  PubMed  Google Scholar 

  22. Odell ID, Cook D (2013) Immunofluorescence techniques. J Invest Dermatol 133:e4

    Article  CAS  PubMed  Google Scholar 

  23. Taylor CR (2013) Chapter 1: Introduction to immunohistochemistry. In: Immunohistochemical Staining Methods Educational IHC Guidebook, 6th edn. Dako, Glostrup, Denmark

    Google Scholar 

  24. Lichtman JW, Conchello JA (2005) Fluorescence microscopy. Nat Methods 2:910–919

    Article  CAS  PubMed  Google Scholar 

  25. Bacallao RSS, Phillips C (2006) Guiding principles of specimen preservation for confocal fluorescence microscopy. In: Handbook Of Biological Confocal Microscopy. Springer, New York, pp 368–380

    Chapter  Google Scholar 

  26. Nakane PK, Pierce GB Jr (1967) Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol 33:307–318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bonsib SM (2002) Differential diagnosis in nephropathology: an immunofluorescence-driven approach. Adv Anat Pathol 9:101–114

    Article  PubMed  Google Scholar 

  28. Qualman SJ, Keren DF (1979) Immunofluorescence of deparaffinized, trypsin-treated renal tissues. Preservation of antigens as an adjunct to diagnosis of disease. Lab Investig 41:483–489

    CAS  PubMed  Google Scholar 

  29. Nasr SH, Fidler ME, Said SM (2018) Paraffin immunofluorescence: a valuable ancillary technique in renal pathology. Kidney Int Rep 3:1260–1266

    Article  PubMed  PubMed Central  Google Scholar 

  30. Nishi K, Isobe S, Zhu Y, Kiyama R (2015) Fluorescence-based bioassays for the detection and evaluation of food materials. Sensors (Basel) 15:25831–25867

    Article  CAS  Google Scholar 

  31. Camp RL, Chung GG, Rimm DL (2002) Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med 8:1323–1327

    Article  CAS  PubMed  Google Scholar 

  32. Zhang W, Sakashita S, Taylor P, Tsao MS, Moran MF (2015) Comprehensive proteome analysis of fresh frozen and optimal cutting temperature (OCT) embedded primary non-small cell lung carcinoma by LC-MS/MS. Methods 81:50–55

    Article  CAS  PubMed  Google Scholar 

  33. Fisher AH, Jacobson KA, Rose J, Zeller R (2008) Cryosectioning Tissues. CSH Protocols 3(8). https://doi.org/10.1101/pdb.prot4991

  34. Donaldson JG (2001) Immunofluorescence staining. Curr Protoc Cell Biol;chapter 4:unit 4 3

    Google Scholar 

  35. Pace GE (2013) Chapter 8: Optimization of immunohistochemical reactions. In: Immunohistochemical Staining Methods Educational IHC Guidebook, 6th edn. Dako, Glostrup, Denmark

    Google Scholar 

  36. Mason DY, Micklem K, Jones M (2000) Double immunofluorescence labelling of routinely processed paraffin sections. J Pathol 191:452–461

    Article  CAS  PubMed  Google Scholar 

  37. van der Loos CM (2008) Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging. J Histochem Cytochem 56:313–328

    Article  PubMed  PubMed Central  Google Scholar 

  38. Mies B (1998) Multiple immunofluorescence microscopy of the cytoskeleton. In: Cell Biology A Laboratory Handbook, 2nd edn. American Press, Boston, MA

    Google Scholar 

  39. Morris RH, Vassalli P, McCluskey RT (1968) Immunofluorescent studies on renal biopsies in pregnancy. Clin Obstet Gynecol 11:522–532

    CAS  PubMed  Google Scholar 

  40. Choi YJ, Reiner L (1980) Immunofluorescence of renal lesions in paraffin-embedded and fresh-frozen sections. Am J Clin Pathol 73:116–119

    Article  CAS  PubMed  Google Scholar 

  41. Michel B, Milner Y, David K (1972) Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases--preliminary report. J Invest Dermatol 59:449–452

    Article  CAS  PubMed  Google Scholar 

  42. Huang SN, Minassian H, More JD (1976) Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Investig 35:383–390

    CAS  PubMed  Google Scholar 

  43. Singh G, Singh L, Ghosh R, Nath D, Dinda AK (2016) Immunofluorescence on paraffin embedded renal biopsies: experience of a tertiary care center with review of literature. World J Nephrol 5:461–470

    Article  PubMed  PubMed Central  Google Scholar 

  44. Larsen CP, Messias NC, Walker PD et al (2015) Membranoproliferative glomerulonephritis with masked monotypic immunoglobulin deposits. Kidney Int 88:867–873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Messias NC, Walker PD, Larsen CP (2015) Paraffin immunofluorescence in the renal pathology laboratory: more than a salvage technique. Mod Pathol 28:854–860

    Article  CAS  PubMed  Google Scholar 

  46. Larsen CP, Boils CL, Cossey LN, Sharma SG, Walker PD (2016) Clinicopathologic features of membranous-like Glomerulopathy with masked IgG kappa deposits. Kidney Int Rep 1:299–305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Fowler CB, Evers DL, O'Leary TJ, Mason JT (2011) Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity. J Histochem Cytochem 59:366–381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Mason JT, O'Leary TJ (1991) Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 39:225–229

    Article  CAS  PubMed  Google Scholar 

  49. Fogazzi GB, Bajetta M, Banfi G, Mihatsch M (1989) Comparison of immunofluorescent findings in kidney after snap-freezing and formalin fixation. Pathol Res Pract 185:225–230

    Article  CAS  PubMed  Google Scholar 

  50. Mubarak M, Kazi Javed I, Kulsoom U, Ishaque M (2012) Detection of immunoglobulins and complement components in formalin fixed and paraffin embedded renal biopsy material by immunoflourescence technique. J Nephropathol 1:91–100

    Article  PubMed  PubMed Central  Google Scholar 

  51. van der Ven K, Nguyen TQ, Goldschmeding R (2007) Immunofluorescence on proteinase XXIV-digested paraffin sections. Kidney Int 72:896

    Article  PubMed  Google Scholar 

  52. Larsen CP, Ambuzs JM, Bonsib SM et al (2014) Membranous-like glomerulopathy with masked IgG kappa deposits. Kidney Int 86:154–161

    Article  CAS  PubMed  Google Scholar 

  53. Nada R, Kumar A, Kumar VG, Gupta KL, Joshi K (2016) Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies. Indian J Nephrol 26:182–187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Shi S, Cheng Q, Zhang P et al (2013) Immunofluorescence with dual microwave retrieval of paraffin-embedded sections in the assessment of human renal biopsy specimens. Am J Clin Pathol 139:71–78

    Article  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Arkana Laboratories, Little Rock, AR, USA

    Vanessa Moreno & Elizabeth A. Smith

  2. Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Sergio Piña-Oviedo

Authors
  1. Vanessa Moreno
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  2. Elizabeth A. Smith
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  3. Sergio Piña-Oviedo
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Corresponding author

Correspondence to Sergio Piña-Oviedo .

Editor information

Editors and Affiliations

  1. Department of Pathology and Medicine & Louisiana Cancer Research Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA

    Luis Del Valle

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Moreno, V., Smith, E.A., Piña-Oviedo, S. (2022). Fluorescent Immunohistochemistry. In: Del Valle, L. (eds) Immunohistochemistry and Immunocytochemistry. Methods in Molecular Biology, vol 2422. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1948-3_9

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  • DOI: https://doi.org/10.1007/978-1-0716-1948-3_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1947-6

  • Online ISBN: 978-1-0716-1948-3

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