Considerations for Immunohistochemistry

  • Swathi Balaji
  • Hui Li
  • Emily Steen
  • Sundeep G. KeswaniEmail author
Part of the Success in Academic Surgery book series (SIAS)


Although immunohistochemistry (IHC) has been known and applied for decades, major advances such as robotic sample processing, digital slide image capture, and innovative computerized data analysis uphold the relevance of the technology in current laboratory practice. In keeping with this notion, the expression of a protein by a given tissue or cell can be readily defined using methods like immunoblotting or mass spectrometry. However, when it comes to assessing the microenvironmental location and potential cellular connections, only IHC can provide the pathophysiological spatial context. Thus, IHC enables the selective localization and visualization of protein antigens in tissue sections by means of antigen-specific antibodies that are conjugated to selective fluorescent or enzymatic tags, which can be readily revealed by fluorescence microscopy or chemically pigmented reactions. Since IHC relies on the highly specific antigen and antibody interactions, the technology can be used to identify cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations and their relevant functional properties, thus underscoring its critical application as a key research/diagnostic tool for investigators studying animal or human tissues. With well-developed tests and controls, correct procedure, and equipment, IHC can be used to analyze a wide variety of cell and tissue structures, processes, and functions, such as cell cycle analysis and tissue protein binding in health and disease. Notably, these applications were previously inaccessible by traditional histochemistry staining techniques, which only identified a limited number of proteins and tissue structures. In the past, this process was semiquantitative at best, but new advances are making it possible to obtain quantitative results using IHC. The present chapter reviews the history and applications of IHC, basic principles, techniques, troubleshooting, analysis, and data interpretation. This chapter will further cover the latest innovations in IHC slide preparation and labeling for advanced diagnostic and prognostic applications, such as CyTOF and laser capture microdissection, which allows high-throughput quantitative methods of proteomics, real-time polymerase chain reaction (qRT-PCR), and genomics on selective cells and tissue regions.


Immunohistochemistry Sample preparation Fixation Embedding Sectioning Antigen retrieval Antibody Staining Counter staining Microdissection Pathology 


  1. 1.
    Marrack JR. Derived antigens as a means of studying the relation of specific combination to chemical structure: (section of therapeutics and pharmacology). Proc R Soc Med. 1934;27(8):1063–5.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Coons AHCH, Jones RN. Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med. 1941;47:200–2.CrossRefGoogle Scholar
  3. 3.
    Nakane PK, Pierce GB Jr. Enzyme-labeled antibodies: preparation and application for the localization of antigens. J Histochem Cytochem. 1966;14(12):929–31.PubMedCrossRefGoogle Scholar
  4. 4.
    Nakane PK. Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J Histochem Cytochem. 1968;16(9):557–60.PubMedCrossRefGoogle Scholar
  5. 5.
    Avrameas S, Uriel J. Method of antigen and antibody labelling with enzymes and its immunodiffusion application. Comptes rendus hebdomadaires des seances de l’Academie des sciences. Serie D: Sciences naturelles. 1966;262(24):2543–5.Google Scholar
  6. 6.
    Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem. 1970;18(5):315–33.PubMedCrossRefGoogle Scholar
  7. 7.
    Mason DY, Sammons R. Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol. 1978;31(5):454–60.PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Mason DY, Sammons R. Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use. J Immunol Methods. 1978;20:317–24.PubMedCrossRefGoogle Scholar
  9. 9.
    Singer SJ. Preparation of an electron-dense antibody conjugate. Nature. 1959;183(4674):1523–4.PubMedCrossRefGoogle Scholar
  10. 10.
    Sternberger LA. Electron microscopic immunocytochemistry: a review. J Histochem Cytochem. 1967;15(3):139–59.PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Faulk WP, Taylor GM. An immunocolloid method for the electron microscope. Immunochemistry. 1971;8(11):1081–3.PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Bullock G, Petrusz P, editors. Techniques in immunocytochemistry. London: Academic; 1982. 4 volumes.Google Scholar
  13. 13.
    Huang SN, Minassian H, More JD. Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Investig. 1976;35(4):383–90.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39(6):741–8.PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29(4):577–80.PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Sabattini E, Bisgaard K, Ascani S, et al. The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol. 1998;51(7):506–11.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Hsu SM, Raine L. Protein A, avidin, and biotin in immunohistochemistry. J Histochem Cytochem. 1981;29(11):1349–53.PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Hsu SM, Raine L, Fanger H. The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Am J Clin Pathol. 1981;75(6):816–21.PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol. 1981;75(5):734–8.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Krishnamurthy VK, Guilak F, Narmoneva DA, Hinton RB. Regional structure-function relationships in mouse aortic valve tissue. J Biomech. 2011;44(1):77–83.PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Gordon A, Kozin ED, Keswani SG, et al. Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation. Wound Repair Regen. 2008;16(1):70–9.PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Balaji S, Vaikunth SS, Lang SA, et al. Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing. Wound Repair Regen. 2012;20(1):15–27.PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Kim SJ, Kim JS, Papadopoulos J, et al. Circulating monocytes expressing CD31: implications for acute and chronic angiogenesis. Am J Pathol. 2009;174(5):1972–80.PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Cho H, Balaji S, Sheikh AQ, et al. Regulation of endothelial cell activation and angiogenesis by injectable peptide nanofibers. Acta Biomater. 2012;8(1):154–64.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Gu B, Kaneko T, Zaw SYM, et al. Macrophage populations show an M1-to-M2 transition in an experimental model of coronal pulp tissue engineering with mesenchymal stem cells. Int Endodont J. 2018. Scholar
  26. 26.
    Das A, Sinha M, Datta S, et al. Monocyte and macrophage plasticity in tissue repair and regeneration. Am J Pathol. 2015;185(10):2596–606.PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Ferraro NM, Dampier W, Weingarten MS, Spiller KL. Deconvolution of heterogeneous wound tissue samples into relative macrophage phenotype composition via models based on gene expression. Integr Biol. 2017;9(4):328–38.CrossRefGoogle Scholar
  28. 28.
    Sicari BM, Dziki JL, Siu BF, Medberry CJ, Dearth CL, Badylak SF. The promotion of a constructive macrophage phenotype by solubilized extracellular matrix. Biomaterials. 2014;35(30):8605–12.PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Lee C, Lee J, Choi SA, et al. M1 macrophage recruitment correlates with worse outcome in SHH Medulloblastomas. BMC Cancer. 2018;18(1):535.PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Minami K, Hiwatashi K, Ueno S, et al. Prognostic significance of CD68, CD163 and folate receptor-beta positive macrophages in hepatocellular carcinoma. Exp Ther Med. 2018;15(5):4465–76.PubMedPubMedCentralGoogle Scholar
  31. 31.
    Yu CC, Woods AL, Levison DA. The assessment of cellular proliferation by immunohistochemistry: a review of currently available methods and their applications. Histochem J. 1992;24(3):121–31.PubMedCrossRefGoogle Scholar
  32. 32.
    Hurley JR, Cho H, Sheikh AQ, et al. Nanofiber Microenvironment Effectively Restores Angiogenic Potential of Diabetic Endothelial Cells. Advances in wound care. 2014;3(11):717–28.PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS, Blomme EA. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol. 2003;199(2):221–8.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Holubec H, Payne CM, Bernstein H, et al. Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa. J Histochem Cytochem. 2005;53(2):229–35.PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Shigenaga MK, Aboujaoude EN, Chen Q, Ames BN. Assays of oxidative DNA damage biomarkers 8-oxo-2′-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 1994;234:16–33.PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Raina AK, Perry G, Nunomura A, Sayre LM, Smith MA. Histochemical and immunocytochemical approaches to the study of oxidative stress. Clin Chem Lab Med. 2000;38(2):93–7.PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Cheuk W, Chan JK. Subcellular localization of immunohistochemical signals: knowledge of the ultrastructural or biologic features of the antigens helps predict the signal localization and proper interpretation of immunostains. Int J Surg Pathol. 2004;12(3):185–206.PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Lewin B. Genes VIII. Pearson Prentice Hall: Upper Saddle River, NJ; 2004.Google Scholar
  39. 39.
    Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Molecular cell. 2007;28(5):730–8.PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Mandell JW. Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol. 2003;163(5):1687–98.PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Baker AF, Dragovich T, Ihle NT, Williams R, Fenoglio-Preiser C, Powis G. Stability of phosphoprotein as a biological marker of tumor signaling. Clinical cancer research. 2005;11(12):4338–40.PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Balaji S, Wang X, King A, et al. Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling. FASEB J. 2017;31(3):868–81.PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Poynter ME, Janssen-Heininger YM, Buder-Hoffmann S, Taatjes DJ, Mossman BT. Measurement of oxidant-induced signal transduction proteins using cell imaging. Free Radic Biol Med. 1999;27(11-12):1164–72.PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Hayes AJ, Hughes CE, Caterson B. Antibodies and immunohistochemistry in extracellular matrix research. Methods. 2008;45(1):10–21.PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Owen GR, Meredith DO, Gwynn I, Richards RG. Focal adhesion quantification - a new assay of material biocompatibility? Review. Eur Cells Mater. 2005;9:85–96. discussion 85–96.PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Evanko SP, Chan CK, Johnson PY, Frevert CW, Wight TN. The biochemistry and immunohistochemistry of versican. Methods Cell Biol. 2018;143:261–79.PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Merrilees MJ, Zuo N, Evanko SP, Day AJ, Wight TN. G1 domain of versican regulates hyaluronan organization and the phenotype of cultured human dermal fibroblasts. J Histochem Cytochem. 2016;64(6):353–63.PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Kelly-Goss MR, Ning B, Bruce AC, et al. Dynamic, heterogeneous endothelial Tie2 expression and capillary blood flow during microvascular remodeling. Sci Rep. 2017;7(1):9049.PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Dyment NA, Kazemi N, Aschbacher-Smith LE, et al. The relationships among spatiotemporal collagen gene expression, histology, and biomechanics following full-length injury in the murine patellar tendon. J Orthop Res. 2012;30(1):28–36.PubMedCrossRefGoogle Scholar
  50. 50.
    Dyment NA, Hagiwara Y, Matthews BG, Li Y, Kalajzic I, Rowe DW. Lineage tracing of resident tendon progenitor cells during growth and natural healing. PLoS One. 2014;9(4):e96113.PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Sinha M, Sen CK, Singh K, et al. Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue. Nat Commun. 2018;9(1):936.PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    True LD. T. Atlas of diagnostic immunohistopathology. Philadelphia, PA: J.B. Lippincott Company; 1990.Google Scholar
  53. 53.
    Malatesta M. Histological and histochemical methods – theory and practice. Eur J Histochem. 2016;60:2639.PubMedCentralGoogle Scholar
  54. 54.
    Microscopy Society of America. Microscopy today. Middleton, WI: Microscopy Society of America; 2018.Google Scholar
  55. 55.
    Nissanov J, Bertrand L, Tretiak O. Cryosectioning distortion reduction using tape support. Microsc Res Tech. 2001;53(3):239–40.PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Sompuram SR, Vani K, Messana E, Bogen SA. A molecular mechanism of formalin fixation and antigen retrieval. Am J Clin Pathol. 2004;121(2):190–9.PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Boenisch T. Heat-induced antigen retrieval: what are we retrieving? J Histochem Cytochem. 2006;54(9):961–4.PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Dabbs DJ. Diagnostic immunohistochemistry. New York: Churchill Livingstone; 2002.Google Scholar
  59. 59.
    Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. Journal of immunological methods. 1989;125(1-2):279–85.PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    van Gijlswijk RP, Zijlmans HJ, Wiegant J, et al. Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem. 1997;45(3):375–82.PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Hatanaka Y, Imaoka Y, Torisu K, et al. A simplified, sensitive immunohistochemical detection system employing signal amplification based on fluorescyl-tyramide/antifluorescein antibody reaction: its application to pathologic testing and research. Appl Immunohistochem Mol Morphol. 2008;16(1):87–93.PubMedPubMedCentralGoogle Scholar
  62. 62.
    Schweitzer B, Wiltshire S, Lambert J, et al. Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci U S A. 2000;97(18):10113–9.PubMedPubMedCentralCrossRefGoogle Scholar
  63. 63.
    Wiltshire S, O’Malley S, Lambert J, et al. Detection of multiple allergen-specific IgEs on microarrays by immunoassay with rolling circle amplification. Clin Chem. 2000;46(12):1990–3.PubMedPubMedCentralGoogle Scholar
  64. 64.
    Sweeney E, Ward TH, Gray N, et al. Quantitative multiplexed quantum dot immunohistochemistry. Biochemical and biophysical research communications. 2008;374(2):181–6.PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Burry RW. Controls for immunocytochemistry: an update. J Histochem Cytochem. 2011;59(1):6–12.PubMedPubMedCentralCrossRefGoogle Scholar
  66. 66.
    Ramos-Vara JA. Principles and methods of immunohistochemistry. Methods Mol Biol. 2011;691:83–96.PubMedCrossRefPubMedCentralGoogle Scholar
  67. 67.
    Fetsch PA, Abati A. The clinical immunohistochemistry laboratory: regulations and troubleshooting guidelines. Methods Mol Biol. 2010;588:399–412.PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    Cregger M, Berger AJ, Rimm DL. Immunohistochemistry and quantitative analysis of protein expression. Arch Pathol Lab Med. 2006;130(7):1026–30.PubMedPubMedCentralGoogle Scholar
  69. 69.
    Taylor CR, Levenson RM. Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006;49(4):411–24.PubMedCrossRefPubMedCentralGoogle Scholar
  70. 70.
    Kononen J, Bubendorf L, Kallioniemi A, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4(7):844–7.PubMedCrossRefGoogle Scholar
  71. 71.
    Kirkeby S, Thomsen CE. Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software. J Immunol Methods. 2005;301(1-2):102–13.PubMedCrossRefGoogle Scholar
  72. 72.
    Schulz KR, Danna EA, Krutzik PO, Nolan GP. Single-cell phospho-protein analysis by flow cytometry. Curr Protoc Immunol. 2012;Chapter 8:Unit 8 17 11–20.Google Scholar
  73. 73.
    Anchang B, Davis KL, Fienberg HG, et al. DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity. Proc Natl Acad Sci U S A. 2018;115(18):E4294–303.PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Fienberg HG, Nolan GP. High-dimensional cytometry. Preface. Curr Topics Microbiol Immunol. 2014;377:vii–viii.Google Scholar
  75. 75.
    Fienberg HG, Nolan GP. Mass cytometry to decipher the mechanism of nongenetic drug resistance in cancer. Curr Top Microbiol Immunol. 2014;377:85–94.PubMedGoogle Scholar
  76. 76.
    Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, Bodenmiller B. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A. 2012;81(6):467–75.PubMedCrossRefPubMedCentralGoogle Scholar
  77. 77.
    Spitzer MH, Nolan GP. Mass cytometry: single cells, many features. Cell. 2016;165(4):780–91.PubMedPubMedCentralCrossRefGoogle Scholar
  78. 78.
    Chang Q, Ornatsky OI, Siddiqui I, Loboda A, Baranov VI, Hedley DW. Imaging mass cytometry. Cytometry A. 2017;91(2):160–9.PubMedCrossRefPubMedCentralGoogle Scholar
  79. 79.
    Datta S, Malhotra L, Dickerson R, Chaffee S, Sen CK, Roy S. Laser capture microdissection: big data from small samples. Histol Histopathol. 2015;30(11):1255–69.PubMedPubMedCentralGoogle Scholar
  80. 80.
    Abdul-Salam VB, Wharton J, Cupitt J, Berryman M, Edwards RJ, Wilkins MR. Proteomic analysis of lung tissues from patients with pulmonary arterial hypertension. Circulation. 2010;122(20):2058–67.PubMedCrossRefPubMedCentralGoogle Scholar
  81. 81.
    O’Rourke MB, Padula MP. Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval. BioTechniques. 2016;60(5):229–38.PubMedPubMedCentralGoogle Scholar
  82. 82.
    Patel PG, Selvarajah S, Boursalie S, et al. Preparation of formalin-fixed paraffin-embedded tissue cores for both RNA and DNA extraction. J Vis Exp. 2016;114
  83. 83.
    Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161(6):1961–71.PubMedPubMedCentralCrossRefGoogle Scholar
  84. 84.
    von Ahlfen S, Missel A, Bendrat K, Schlumpberger M. Determinants of RNA quality from FFPE samples. PloS one. 2007;2(12):e1261.CrossRefGoogle Scholar
  85. 85.
    Graw S, Meier R, Minn K, et al. Robust gene expression and mutation analyses of RNA-sequencing of formalin-fixed diagnostic tumor samples. Sci Rep. 2015;5:12335.PubMedPubMedCentralCrossRefGoogle Scholar
  86. 86.
    Santos MC, Saito CP, Line SR. Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods. Pathol Res Pract. 2008;204(9):633–6.PubMedCrossRefPubMedCentralGoogle Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Swathi Balaji
    • 1
  • Hui Li
    • 1
  • Emily Steen
    • 1
  • Sundeep G. Keswani
    • 1
    Email author
  1. 1.Laboratory for Regenerative Tissue Repair, Division of Pediatric Surgery, Department of SurgeryBaylor College of Medicine and Texas Children’s HospitalHoustonUSA

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