Immunohistochemistry: Fundamentals and Applications in Oncology

José A. Ramos‐Vara1 and Luke B. Borst2

1Purdue University, USA

2North Carolina State University, USA


In many laboratories, immunohistochemistry (IHC) has become a routine supplement to the classic morphologic approach of investigational pathology.1 Though its main application in veterinary oncology is in the characterization and diagnosis of neoplastic diseases, current trends highlight a central role for IHC in prognosis and theranostics.2–4

IHC bridges three disciplines: immunology, histology, and chemistry. Antigens are recognized in histologic sections by specific antibodies. The antigen–antibody binding is then visualized by light or fluorescent microscopy as a colored histochemical reaction.5 The value of IHC in the study of neoplastic diseases has been enhanced by increased sensitivity and specificity, improved detection of multiple antigens in the same histologic section, simultaneous evaluation of numerous samples with tissue microarrays, new antigen retrieval (AR) methods, and sophisticated automation.6

The authors believe IHC is a powerful tool in oncology, but only in concert with morphology. A blue, red, or brown reaction with IHC is of little or no help if not accompanied by an understanding of the morphologic changes typical of a specific neoplasm. Therefore, IHC is best utilized as a complement to morphology and should be considered as a piece of data in the diagnostic puzzle. IHC used in isolation or as the test to overrule all others may lead to spurious results. As Basturk et al. stated: “There is no IHC marker that makes the diagnosis by itself. Exceptions always occur, and unfortunately, they tend to merge when IHC is necessary the most”.7 For a review of immunocytochemistry applied to neoplastic diseases, please see reference 8.


  1. 1. Ramos‐Vara, J.A., Kiupel, M., and Miller, M.A. (2005) Veterinary diagnostic immunohistochemistry: a survey of 47 laboratories. J Histotechnol 28:19–23.
  2. 2. Kiupel, M., Webster, J.D., Kaneene, J.B., et al. (2004) The use of KIT and tryptase expression patterns as prognostic tools for canine cutaneous mast cell tumors. Vet Pathol 41:371–377.
  3. 3. Leong, A.S.‐Y. and Leong, T. Y.‐M. (2006) Newer developments in immunohistology. J Clin Pathol 59:1117–1126.
  4. 4. Teruya‐Feldstein, J. (2010) The immunohistochemistry laboratory. Looking at molecules and preparing for tomorrow. Arch Pathol Lab Med 134:1659–1665.
  5. 5. Ramos‐Vara, J.A. (2005) Technical aspects of immunohistochemistry. Vet Pathol 42:405–426.
  6. 6. Prichard, J.W. (2014) Overview of automated immunohistochemistry. Arch Pathol Lab Med 138:1578–1582.
  7. 7. Basturk, O., Farris III, A.B., and Adsay, N.V. (2010) Immunohistology of the pancreas, biliary tract, and liver. In Diagnostic Immunohistochemistry. Theranostic and Genomic Applications, 3rd edn. (ed. D.J. Dabbs). Saunders, St. Louis, MO, pp. 541–592.
  8. 8. Ramos‐Vara, J.A., Avery, P.R., and Avery, A.C. (2015) Advanced diagnostic techniques. In Canine and Feline Cytology: A Color Atlas and Interpretation Guide, 3rd edn. (eds. R.E. Raskin and D. Meyer). Elsevier, St. Louis, MO, pp. 453–494.

The immunohistochemical test

The IHC technique is a combination of immunologic and chemical reactions visualized with a photonic microscope. The technique can be divided into three main phases (Table 3.1). Phase 1 (preanalytical) starts with sample procurement, followed by tissue fixation, trimming, embedding, and tissue sectioning on a microtome. Phase 2 (analytical) starts with deparaffination of tissue sections and includes all the steps from AR and blocking nonspecific activities through the binding and dection of primary antibody and ending with counterstaining and coverslipping. Phase 3 (postanalytical) includes evaluation of the IHC control, interpretation of results and generation of an IHC report.1

Table 3.1 Steps in an immunohistochemical test

Steps Variables
Preanalytical phase
Sample procurement Delayed fixation, prolonged ischemia, thickness of sample
Fixation Cross‐linking vs. coagulating fixatives, duration
Decalcification Type of decalcification solution and duration
Tissue processing Paraffin‐embedded vs. frozen tissues
Tissue sectioning Thickness of tissue section; drying temperature and duration; tissue section aging
Analytical phase
Deparaffination Dewaxing agent
Antigen retrieval Detergents, enzymes, HIER
Blocking nonspecific reactivities Endogenous enzymes, hydrophobic binding, pigments
Primary antibody Monoclonal vs. polyclonal; Ag recognition (native vs. linear); specificity; species variability
Detection system Avidin–biotin vs. polymer‐based systems; ultrasensitive methods
Enzyme–substrate–chromogen Color detection
Multiplex IHC Enzyme–substrate combinations
Counterstain Contrast between chromogen and counterstain
Postanalytical phase
Control performance Animal species compatibility, tissue processing
Interpretation Pathologist vs. automated evaluation
Report Percentage of positive cells; positive vs. negative threshold; standalone test vs.ancillary test

Diagnostic, prognostic or theranostic test

Fixation and processing


Fixation of tissues is necessary to (1) preserve cellular components (e.g., including soluble and structural proteins); (2) prevent autolysis and displacement of cell constituents (e.g., antigens and enzymes); (3) stabilize cellular materials against deleterious effects of subsequent procedures; and (4) facilitate conventional staining and immunostaining.2 However, there is no “one fixative fits all” in IHC; in fact, some antigens can only be detected in fresh frozen sections.3 In general, two types of chemical fixatives are used in histopathology: cross‐linking (noncoagulating) fixatives and coagulating fixatives.4

Formaldehyde is a cross‐linking fixative and is the standard and most common fixative for both routine histology and IHC. Formaldehyde preserves mainly peptides and the general structure of cellular organelles. It also interacts with nucleic acids, but has minimal preservation of carbohydrates.5,6 It can be a good preservative of lipids if the fixative is supplemented with calcium.7 Formaldehyde is used mainly because it is reliable for general histology, it is inexpensive, and its deleterious effects can often be countered with AR. In addition, over the decades pathologists have become accustomed to the artifacts produced by this fixative.8 In contrast, formalin‐substitute fixatives are often based on alcohol solutions; therefore, their mechanism of fixation is dehydration and protein coagulation.9–12 A recent study indicates that Weigners fixative (a mixture of alcohols and pickling salt) performs comparably to formaldehyde for IHC and even maintained immunoreactivity for some biomarkers after prolonged fixation, whereas formaldehyde did not.13 If a formalin substitute is used for an IHC test already validated on formalin‐fixed tissues, a new validation of the test is mandatory.14

Fixation with formaldehyde is a time‐dependent, three‐step process that entails penetration, covalent bonding (binding), and formation of cross‐links.10,15 These steps happen simultaneously, but at very different rates, with penetration occurring around 12 times faster than binding, and binding 4 times faster than cross‐linking.10 As an example, a 3‐mm‐thick sample will be 100% penetrated, 24% bonded and 6% cross‐linked in 8 hours; after 24 hours of fixation it will be 70% bonded and 36% cross‐linked.16,17 Fixation is also temperature dependent; fixation at 37 °C occurs faster than at 25 °C.18 Although there is no optimal standard fixation time for every antigen, it is recommended that diagnostic specimens be fixed for 16–32 hours, with complete fixation, on average, achieved after 24–48 hours.14 Samples to be fixed should not be thicker than 2–4 mm.

The basic mechanism of fixation with formaldehyde is the formation of addition products (adducts) between the formalin and uncharged reactive amino groups (‐NH or ‐NH2), which eventually will form cross‐links.18 Once the addition product (reactive hydroxymethyl compound) is formed, additional cross‐linking will happen. In the presence of a second reactive hydrogen, the hydroxymethyl group will form a methylene bridge. Therefore, the final result of formaldehyde fixation is a profound change in the conformation of macromolecules, which may make the recognition of antigens by antibodies impossible or, at best, difficult (Figure 3.1).19 For example, these changes modify the three‐dimensional (tertiary and quaternary) structure of proteins, whereas the primary and secondary structures are little affected.2,20 Changes in the tertiary structure of proteins may also occur in subsequent interactions of cross‐linked proteins with ethanol or clearing agents during tissue processing.15,21,22

Simplified schematic flow diagram of the effects of formalin fixation on proteins/

Figure 3.1 Effects of formalin fixation on proteins. Formalin fixation produces conformational changes in proteins secondary to cross‐links between protein groups and the fixative. The use of antigen retrieval (heat‐induced epitope retrieval, HIER) is intended to revert those changes.

(Source: Ramos‐Vara and Miller, 2014. Reproduced with permission of SAGE Publications.)

The fixation step is a critical quality control point, so standardized laboratory practices are needed to ensure accurate and repeatable IHC using formalin‐fixed paraffin‐embedded (FFPE) tissues.14 Prolonged fixation thoretically can produce false‐negative results due to excessive cross‐links;2 however, the use of heat‐based AR methods considerably mitigates the impact of overfixation in many cases (Figure 3.1).23–30 That said, the effect of prolonged fixation with formaldehyde on an antigen located in different cell compartments may vary (Figure 3.2).2

Micrographs of a horse fetus lung infected with Bartonella sp. (top panels) and dog skin with cytokeratins (bottom panels).

Figure 3.2 Effects of fixation on antigen expression. Antigens are affected by fixation differently. (A,B) Horse, fetus lung infected with Bartonella sp. (A) Fixation for 2 days: antigen (arrowheads) is easily detected. (B) Fixation for 11 weeks: Antigen detection is lost. (C,D) Dog, skin. Cytokeratins. (C) Fixation for 2 days: Strong detection in adnexal structures. (D) Fixation for 7 weeks: Significant loss of immunoreactivity. Immunoperoxidase‐DAB.

(Source: Ramos‐Vara and Miller, 2014. Reproduced with permission of SAGE Publications.)

Underfixation is currently considered a more common and serious problem than overfixation.14,31 If large samples are underfixed, cross‐links develop only in the periphery of the specimen with the core of the tissue left unfixed or fixed through coagulation by the series of alcohol gradients prior to paraffin embedding.2,32 This can result in a staining gradient in IHC that can confound interpretation (Figure 3.3).14 In addition, underfixed tissues may undergo inadequate antigen retrieval or produce unexpected antigen detection, which can result in false‐negative or false‐positive results, respectively.33,34

Micrograph of a sample of a lymph node in a dog with reduced hematoxylin staining at the center (left) and with a lack of staining for CD79 in the center (right).

Figure 3.3 Effects of inadequate fixation. Dog, lymph node. Diffuse large B cell lymphoma. This sample was submitted without slicing, resulting in incomplete penetration of fixative and inadequate staining. (A) Reduced staining with hematoxylin in the center of the sample. (B) Lack of staining for CD79a in the center of the sample. Immunoperoxidase‐DAB.

(Source: Ramos‐Vara and Miller, 2014. Reproduced with permission of SAGE Publications.)

Like underfixation, delayed fixation may affect IHC results, particularly when apoptotic markers or markers recognizing phosphorylated proteins are used.14,35–37 Enzyme‐rich tissues, such as intestine or pancreas, autolyze rapidly; proteolysis may lead to increased background staining.14 Diffusion of soluble proteins may occur with fixation delay and has been reported with thyroglobulin, myoglobin, glial fibrillary acid protein (GFAP), and other cellular proteins.38–40


Decalcification with weak acids does not seem to interfere significantly with IHC for most antigens, provided the tissues are well fixed in formalin.32,41 Strong acid decalcifying solutions have negative effects on immunoreactivity, at least for some antigens.42–45 Therefore, weak acid (e.g., formic acid) decalcifying solutions diluted in formalin are recommended for IHC. Due to their potential negative effects, if decalcifying solutions are required then the IHC report should indicate the type of decalcification.

Tissue processing and incubation buffers

Fixation may not be the sole cause of failure to detect an antigen; in some cases, tissue processing is also a key limiting factor.46–48 Evidence for a cumulative effect of fixation and tissue processing as the major cause of lack of antigen recognition in FFPE tissues has been reported.10,49 Changes in the tertiary structure of proteins during the dehydration and clearing steps of tissue processing can reduce or abolish antibody recognition without AR or can increase background staining.14 These negative effects depend on the dehydrating and clearing agent used.50

Paraffin section drying and storage of paraffin blocks

Paraffin sections dried overnight at temperatures of 60 °C or higher may have reduced immunoreactivity for some antigens.50–53 However, a combination of temperature and duration of drying may be more influential than temperature alone.54

Paraffin blocks contain archived tissue suitable to evaluate morphologic and immunohistochemical features of diseases and apply the results to targeted medicine.55 It is the opinion of the authors and others that paraffin blocks remain stable for years in terms of antigenicity.50 Any deleterious effects of prolonged paraffin block storage on tissue antigenicity could differ among antigens and should be considered if IHC results on old paraffin blocks are inconsistent or unexpected.

Storage of unstained paraffin control tissue sections increases efficiency but may adversely affect immunoreactivity (tissue section aging/slide oxidation/biomolecule degradation).14,50,56 These differences are epitope specific rather than protein target specific.14 Both light (photo‐oxidation) and temperature contribute to this process.4 Tissue section aging is a common problem with nuclear antigens (e.g., Ki67, estrogen receptor, p53); however, in a recent study with markers employed in animal tissues, cytoplasmic membrane antigens were more sensitive than nuclear antigens to degradation from photo‐oxidation and/or ambient temperature.4,33,57–66 Others have reported a negative effect on immunoreactivity when using tissues suboptimally dehydrated (retained endogenous water) during paraffin embedding or with paraffin sections stored under high humidity conditions.67 To reduce antigen degradation due to oxidation, paraffin sections should be used within a week of sectioning or at least should be stored in an airtight container in the dark and in the cold (refrigerator or freezer).4,14


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Antigen retrieval

Because fixation and tissue processing modify the three‐dimensional structure of proteins (antigens), these essential steps can render certain antigens undetectable by specific antibodies. This dilemma is better understood if we remember that, in general, an immunologic reaction between antigen and antibody depends on the conformation of the former.1 Antigen retrieval (AR) methods that reverse the conformational changes in FFPE tissues are particularly important (Figure 3.1). Approximately 85% of antigens fixed in formalin require some type of AR to optimize the immunoreaction.2 The two most common AR procedures in IHC are enzymatic and heat‐based retrieval.

Enzymatic retrieval

Protease‐induced epitope retrieval (PIER) was the most commonly used AR method before the advent of heat‐based AR in the 1990s. Many enzymes (e.g., trypsin, proteinase, K., pronase, ficin, and pepsin) have been used for AR.3–7 The mechanism of PIER is probably protein digestion, but the cleavage is nonspecific and some antigens may be negatively affected.8 It is our preference to optimize a few enzymes rather than to use a broad range of enzymes. The disadvantages of PIER are that it is the optimal AR method for only a few antigens, that it can alter tissue morphology, and that it can damage epitopes.6,8

Heat‐induced epitope retrieval (HIER)

HIER was introduced by Shi et al. and is based on a concept developed by Fraenklen‐Conrat et al., who documented that the chemical reactions between proteins and formalin may be reversed, at least in part, by high‐temperature heating or strong alkaline hydrolysis.9–12 HIER involves heating FFPE tissue sections in an AR buffer. The mechanism involved in HIER is not completely understood, but its final effect is reversal of changes produced during fixation. The mechanism requires the dissociation of irrelevant proteins from target peptides.13 The secondary and tertiary structure of proteins is probably modified (denatured) during HIER; however, this does not affect the immunoreactivity of most antigens because IHC antibodies require only an intact primary (linear) protein structure.14,15 Tissue‐bound calcium ions may be important in masking some antigens during fixation, explaining why calcium chelating substances (e.g., EDTA) are sometimes more effective than citrate buffer in antigen retrieval.16–19 Restoring native electrostatic charges modified during formalin fixation has also been considered as an AR mechanism.20,21

Various types of equipment may be used for HIER, including a de‐cloaker (commercial pressure cooker with electronic controls for temperature and time), vegetable steamer, water bath, microwave oven, or pressure cooker.9,18,22–24

A universal AR solution is not available. AR solutions which have been used in HIER vary in the composition of the buffer (e.g., citrate, tris, Tris‐HCl) and pH range (3–10).1,25 Successful AR retrieval using HIER requires the optimum combination of incubation temperature and AR solution pH and chemical composition. For example, some antigens can be retrieved with low pH solutions, others only with high pH solutions, and a third group with solutions with a wide pH range.26,27 In our and others’ experience, HIER with 0.01 M sodium citrate buffer (pH 6.0) frequently results in successful AR, while preserving cell morphology better than higher pH buffers or solutions containing EDTA.1,28,29 However, the possibility of unexpected immunoreactivity (false‐positive labeling) should always be considered when using HIER, particularly with low pH buffers.28

Recently, so‐called three‐in‐one retrieval buffers have been introduced for use in IHC. These buffers can perform aqueous deparaffination, rehydration, and AR.30 However, their use in IHC is relatively new, and comparison with traditional means of deparaffination, rehydration, and AR is required before routine use in a diagnostic setting as issues related to incomplete removal of paraffin by these buffers have been reported.31

The use of mechanical vibrations by ultrasound as an AR method has been successful in restoring tissue immunoreactivity for some antigens.32–34 This method presumably breaks cross‐links produced during fixation.


  1. 1. Hayat, M.A. (2002) Factors affecting antigen retrieval. In Microscopy, Immunohistochemistry, and Antigen Retrieval Methods for Light and Electron Microscopy (ed. M.A. Hayat). Kluwer Academic, Dordrecht, pp. 53–69.
  2. 2. Ramos‐Vara, J.A. and Beissenherz M. (2000) Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin‐fixed, paraffin‐embedded tissues: experience with 63 markers. J Vet Diagn Invest 12:307–311.
  3. 3. Battifora, H., Kopinski M. (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. J Histochem Cytochem 34:1095–1100.
  4. 4. Jacobsen, M., Clausen, P.P., Smidth S. (1980) The effect of fixation and trypsinization on the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material. Acta Path Microbiol Scand A. 88:369–376.
  5. 5. Miettinen M. (1989) Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity. Path Res Pract.184:431–436.
  6. 6. Ordóñez, N.G., Manning, J.T., Brooks, T.E. (1988) Effect of trypsinization on the immunostaining of formalin‐fixed, paraffin‐embedded tissues. Am J Surg Pathol 12:121–129.
  7. 7. Pinkus, G.S., O’Connor, E.M., Etheridge, C.L., Corson, J.M. (1985) Optimal immunoreactivity of keratin proteins in formalin‐fixed, paraffin‐embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumors using polyclonal and monoclonal antibodies. J Histochem Cytochem.33:465–473.
  8. 8. Van Hecke D. (2002) Routine immunohistochemical staining today: choices to make, challenges to take. J Histotechnol. 25:45–54.
  9. 9. Shi S‐R, Key, M.E., Kalra, K.L. (1991) Antigen retrieval in formalin‐fixed, paraffin‐embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 39:741–748.
  10. 10. Fraenkel‐Conrat, H., Brandon, B.A., Olcott, H.S. (1947) The reaction of formaldehyde with proteins. IV. Participation of indole groups. Gramicidin. J Biol Chem. 168: 99–118.
  11. 11. Fraenkel‐Conrat, H., Olcott, H.S. (1948a) The reaction of formaldehyde with proteins. V. Cross‐linking between amino and primary amide or guanidyl groups. J Am Chem Soc. 70:2673–2684.
  12. 12. Fraenkel‐Conrat, H., Olcott, H.S. (1948b) Reaction of formaldehyde with proteins. VI. Cross‐linking of amino groups with phenol, imidazole, or indole groups. J Biol Chem. 174:827–843.
  13. 13. Buchwallow, I.B., Böcker W. (2010) Immunostaining enhancement. In: Buchwallow, I., Böcker, W., eds. Immunohistochemistry: Basics and Methods.Springer Verlag, pp.47–59.
  14. 14. Bogen, S.A., Vani, K., Sompuram, S.R. (2009) Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin‐induced cross‐links. Biotech Histochem. 84:207–215.
  15. 15. Sompuram, S.R., Vani, K., Bogen, S.A. (2006) A molecular model of antigen retrieval using a peptide array. Am J Clin Pathol 125:91–98.
  16. 16. Morgan, J.M., Navabi, H., Schmid, K.W., et al. (1994) Possible role of tissue‐bound calcium ions in citrate‐mediated high‐temperature antigen retrieval. J Pathol 174:301–307.
  17. 17. Morgan, J.M., Navabi, H., and Jasani, B. (1997) Role of calcium chelation in high‐temperature antigen retrieval at different pH values. J Pathol 182:233–237.
  18. 18. Pileri, S.A., Roncador, G., Ceccarelli, C., et al. (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116–123.
  19. 19. Wilson, J.E. (1991) The use of monoclonal antibodies and limited proteolysis in elucidation of structure‐function relationships in proteins. Meth Biochem Anal 35:207–250.
  20. 20. Boenisch, T. (2002) Heat‐induced antigen retrieval restores electrostatic forces: prolonging the antibody incubation as an alternative. Appl Immunohistochem Mol Morphol 10:363–367.
  21. 21. Boenisch, T. (2006) Heat‐induced antigen retrieval: what are we retrieving? J Histochem Cytochem 54:961–964.
  22. 22. Bankfalvi, A., Navabi, H., Bier, B., et al. (1994) Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry. J Pathol 174:223–228.
  23. 23. Elias, J.M. (2001) Antigen restoration. The “hot” revolution in immunohistochemistry. J Histotechnol 24:193–198.
  24. 24. Kanai, K., Nunoya, T., Shibuya, K., and Tajima, M. (1998) Variation in effectiveness of antigen retrieval pretreatment for diagnostic immunohistochemistry. Res Vet Sci 64:57–61.
  25. 25. Imam, S.A., Young, L., Chaiwun, B., and Taylor, C.R. (1995) Comparison of two microwave based antigen‐retrieval solutions in unmasking epitopes in formalin‐fixed tissue for immunostaining. Anticancer Res 15:1153–1158.
  26. 26. Shi, S.‐R., Imam, S., Young, L., et al. (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43:193–201.
  27. 27. Shi, S.‐R., Cote, R.J., and Taylor, C.R. (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49:931–937.
  28. 28. Battifora, H. (1999) Quality assurance issues in immunohistochemistry. J Histotechnol 22:169–175.
  29. 29. Ehara, H., Deguchi, T., Koji, T., et al. (1996) Autoclave antigen retrieval technique for immunohistochemical staining of androgen receptor in formalin‐fixed paraffin sections of human prostate. Acta Histochem Cytochem 29:311–318.
  30. 30. Hewitt, S.M., Robinowitz, M., Bogen, S.A., et al. (2011) Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline, 2nd edn. CLSI document I/LA28‐A2. Clinical and Laboratory Standards Institute, Wayne, PA.
  31. 31. Freeland, J., von Bueren, E., Muralitharan, S., et al. (2013) Water‐soluble organic solvent improves paraffin displacement in all‐in‐one epitope retrieval buffers. J Histotechnol 36:51–58.
  32. 32. Gimeno, E., Massone, A., and Portiansky E. (1998) Preembedding epitope retrieval. An ultrasound‐based method for unmasking desmin in tissue blocks. Appl Immunohistochem 6:35–41.
  33. 33. Portiansky, E. and Gimeno E. (1996) A new epitope retrieval method for the detection of structural cytokeratins in the bovine prostatic tissue. Appl Immunohsitochem 4:208–214.
  34. 34. Portiansky, E., Massone, A., and Gimeno E. (1997) Kinetics of epitope retrieval techniques for unmasking cytokeratins in bovine prostatic tissues after different formaldehyde fixation timers. Appl Immunohistochem 5:194–201.

Antigens and antibodies

Fundamental to the IHC test is the ability of an antibody to specifically recognize and strongly bind to an epitope on the desired target antigen.1 From a biochemical point of view, these antigen–antibody (Ag–Ab) interactions are the result of noncovalent bonds and are caused primarily by hydrophobic, electrostatic, and van der Waals forces.2 The specificity of the IHC test is largely dependent on the the ability of the primary antibody to bind to epitopes on the target antigen without cross‐reacting to epitopes on off‐target antigens.


Regions of an antigen that are recognized and bound by antibodies are called epitopes and generally consist of 5 or 6 amino acid residues.1 Multivalent antigens have multiple epitopes which can be either identical (homopolymeric) or unique (heteropolymeric).3 In addition, a specific antigen can have different structures (isoforms) which can contain unique epitopes.3 For example, most protein antigens are multivalent and heteropolymeric which means that for a specific protein antigen, several different antibodies can be raised that bind unique epitopes with variable strength (affinity). Furthermore, these antibodies may or may not recognize an isoform of the same protein. Two broad groups of immunogens are used to produce antibodies: synthetic peptides and purified proteins.4 Synthetic peptides have the advantage of a known amino acid sequence. However, synthetic peptides may lack the normal three‐dimensional structure of the native protein, and other proteins can be intimately associated with the protein of interest in vivo. Both these factors can mask target epitopes, preventing their detection by antibodies raised to synthetic peptides. Also in vivo post‐translational modifications of the native antigen are not present in synthetic peptides.5,6 These factors theoretically decrease the sensitivity of primary antibodies raised to synthetic peptides for target antigens. Furthermore, using synthetic peptides as immunogens can result in decreased antibody specificity. For example, the sequence of the synthetic peptide could also be present in unrelated antigens, producing immunologically specific antigen–antibody binding (molecular mimicry) despite the absence of the target antigen.7–9 Use of purified proteins as immunogens avoids many of the problems of synthetic peptides.4 However, purification of a protein to homogeneity from either cells or tissues can be technically difficult. Also, contaminating proteins may be more antigenic than the protein of interest, producing a disproportionate and unwanted immunogenic response. Another problem with purified proteins arises if the targeted antigen includes highly immunogenic epitopes that are not specific to the antigen of interest.


For the IHC test, the most commonly used immunoglobulin (Ig) is IgG; IgM is used less commonly.9 Immunoglobulins are “Y” shaped and consist of two identical light chains and two identical heavy chains (Figure 3.4). The heavy chains determine the antibody class. The tail of the Y is called Fc (fragment, crystallizable) and is composed of two heavy chains on the C‐terminal side.10 Each end of the forked portion of the Y is called Fab (fragment, antigen‐binding) region. The light chains of most vertebrate antibodies have two distinct forms called kappa and lambda. In any immunoglobulin molecule, both light chains and both heavy chains are of the same type. The light chains consist of two distinct regions: the C‐terminal half of the chain is constant and called CL (constant: light chain), whereas the N‐terminal half of the chain has abundant sequence variability and is called the VL (variable: light chain) region. The Fab (antigen‐binding) region of the immunoglobulin has variable and constant segments of the heavy and light chains. The Fc portion determines the biological functions and permits antibody binding to other antibodies, complement, and immune cells with Fc receptors.9 The specific binding of an antibody to an antigen occurs via hypervariable regions of both heavy and light chains of the amino terminus.9 The antigen‐binding site of an antibody is called the paratope.11

Structure of immunoglobulin molecule with arrows labeling the antigen-binding in the variable region, light and heavy chains, antibody-binding in Fc fragment, and the F(ab) fragment.

Figure 3.4 Structure of an immunoglobulin molecule. In immunohistochemistry, both the variable (antigen‐binding site) and the Fc (antibody‐binding site) regions are necessary for proper detection of the antigen–antibody reaction.

(Source: Ramos‐Vara and Miller, 2014. Reproduced with permission of SAGE Publications.)

Paratopes recognize either linear or conformational epitopes.12 Linear epitopes are a group of 5–7 (and up to 21) contiguous amino acids. Conformational (discontinuous) epitopes, the typical form, consist of small groups of amino acids brought together by conformational folding or binding.13 Although most epitopes involved in immune responses are believed to be conformational, there is evidence that antibodies used in FFPE tissues mainly, or exclusively, recognize linear epitopes.12,13


Affinity is a thermodynamic expression of the binding strength of an antibody (paratope) to an antigenic determinant (epitope).3 Affinity can be defined in mathematical terms as an affinity constant (Ka), which represents the amount of antibody–antigen complex that will be formed at equilibrium. The affinity of the antigen–antibody reaction has practical importance in that high‐affinity antibodies can (1) bind more antigen in a shorter incubation time than low‐affinity antibodies; and (2) in general, be used at lower concentrations than low‐affinity antibodies.14


Avidity (also called functional affinity) is a measure of strength of binding between bi‐ or multivalent antigens and antibodies (combined bond affinities).1 Avidity is the result of the affinity(ies) of the antibody for the epitope(s), the number of antibody‐binding sites, and the geometry of the antigen–antibody complexes.3 As a result, an antibody of IgM (decavalent) has a higher avidity (although typically lower affinity) than an antibody of IgG (bivalent).1,3

Polyclonal antibodies

Polyclonal antibodies (PAbs) are produced with purified antigen in various species (mouse, rabbit, goat, donkey, horse, hamster, sheep, guinea pig, rat, chicken); however, the rabbit is the most common species. PAbs have higher affinity and wide reactivity, but lower specificity in comparison to monoclonal antibodies (mAbs).11 Polyclonal antisera include not only several different antibodies to multiple valencies on the target protein, but also irrelevant antibodies that can be present in high concentration (up to 10 mg/mL) if not affinity‐purified.4,15 PAbs have the advantage over mAbs in that they often identify multiple epitopes and isoforms of the target protein. However, variations in antibody titer and quality, depending on the animal immunized, also contribute to variance among batches of antibodies.16 The “immunologic promiscuity” of PAbs, which can be an advantage (e.g., more possibilities of detecting an antigen due to multiplicity of epitope recognition) can also be a disadvantage: The greater the number of different antibodies to the target protein in the preparation, the greater the likelihood of cross‐reactivity with similar epitopes in other proteins and, therefore, the greater the likelihood of false‐positives.4

Monoclonal antibodies

Monoclonal antibodies (MAbs) are produced mostly in mice and react with only one valency site per antigen.17 Background reactivity with MAbs due to nonspecific Igs is reduced (ascites fluid) or non‐existent (cell culture supernatant). The high specificity reduces, but does not eliminate, the possibility of cross‐reactivity with other antigens because MAbs target epitopes consisting of a small number of amino acids, which can be contained in multiple proteins and peptides.18

MAbs can also be produced in rabbits. The advantages of rabbit over mouse MAbs are their higher affinity (probably a result of high glycosylation), suitability for use on mouse tissues without special procedures, increased specificity in some cases, and less need for antigen retrieval.1,19–23 In a study comparing 10 rabbit MAbs with 10 mouse MAbs targeting the same protein in animal tissues, rabbit MAbs were superior for some markers; however, there was no significant overall improvement in immunoreactivity.24

What makes an antibody good for immunohistochemistry?

A reliable IHC test must be both specific and sensitive; however, there is often confusion surrounding the application of these terms. The confusion stems from the fact that these terms can be applied both to the primary antibody itself as well as to the IHC test as a whole. Further blurring the distinction between antibody and test is the fact that the specificity of the IHC test in large part derives from the primary antibody’s ability to recognize a specific antigen. Likewise, the sensitivity of the IHC test derives from a combination of the binding properties of the primary antibody and the subsequent detection methods. Given the possibilities for confusion, it may be useful to introduce more specific terminology to distinguish the sensitivity and specificity characteristics of the primary antibody from the IHC test as a whole.

For example, molecular specificity is the ability of a primary antibody to bind strongly with a specific epitope of the target antigen; this depends on the antibody molecular structure.1 In oncology, ideal molecular specificity would be an antibody that recognizes one antigen only in tumors of one cell type. There are few if any such antibodies. While many primary antibodies specifically recognize their target antigen, these antigens frequently can be expressed by multiple cell types or are expressed by normal cells of a certain type. All markers also have the potential to be expressed by a tumor line that is not expected to have that antigen. As an example, MUM1 is used to recognize plasma cell tumors (B cell differentiation) because it is required for immunoglobulin light‐chain rearrangement at the pre‐B stage of lymphocyte maturation. MUM1 is expressed in the majority of (but not all) cutaneous plasmacytomas and multiple myelomas. In humans, the molecular specificity of MUM1 is diminished because it is expressed in melanoma and T cell lymphomas, including adult T cell leukemia lymphoma and anaplastic large cell lymphoma. However, in dogs MUM1 has excellent molecular specificity as it is not expressed in common differentials for plasma cell tumors, including melanoma, mast cell tumors, histiocytomas, cutaneous and systemic histiocytoses, histiocytic sarcomas, epitheliotroic lymphomas, and T cell lymphomas.25 Furthermore, target antigens expressed by normal cells of a certain type may be variably expressed (downregulated or upregulated) in neoplastic populations. Therefore it is clear that these variations in molecular specificity impact the diagnostic specificity and sensitivity of the IHC test.

Likewise, the analytical sensitivity of the IHC test depends, in part, on the primary antibody and is defined as the ability of the test to detect low amounts of antigen over background. Analytical sensitivity is a function of the binding characterists of the primary antibody and the detection methods. Polyclonal antibodies may have more analytical sensitivity than mAbs because of polyvalent binding.23 Polymeric labeling and catalyzed signal amplification detection methods can increase analytical sensitivity by amplifying labeling.1 Detection of low amounts of target antigen (analytical sensitity) is critical to the overall sensitivity and specificity of the IHC.

Probably of greatest interest to the diagnostic pathologist is the diagnostic specificity and sensitivity of an IHC test. These are calculated during test standardization and validation (see below) by comparing IHC detection of target antigen in a reference set of “known” normal tissue and tumor types, which are usually determined by morphology.26 However, the approach of using morphology as the gold standard when calculating diagnostic sensitivity and specificity can only provide an approximation of the true diagnostic sensitivity and specificity. Inevitably, tumors of a certain type may exhibit unexpected labeling or indiscernable morphology. As mentioned, the latter case is when IHC would be the most useful; however, these types of tumors are not frequently included during test validation for obvious reasons. As the molecular pathogenesis of specific neoplasms is elucidated, IHC tests might be validated using a combination of morphology and genetic markers that would allow inclusion of these problematic cases.

One approach to increase the diagnostic specificity and sensitivity of the IHC test is the application of mixtures of primary antibodies as a “cocktail.” These cocktails exploit the molecular specificity and analytical sensitivity of multiple antibodies to target a specific molecule or cell type. A well‐known example is pancytokeratin. This antibody mixture contains multiple antibodies with specificity to one or more of the 20 keratins that occur in different epithelia (see discussion of Cancer of unknown primary site below). This extremely useful antibody cocktail sensitively detects the keratins in all epithelia via the molecular specificity of the component antibodies. Alternatively, antibody cocktails have been developed to accurately label specific tumor types. A recent example is the development of a cocktail of antibodies for the diagnosis of amelanotic melanoma in dogs.27 In this approach, multiple antibodies that target different cellular components of melanoma cells are combined to increase the diagnostic sensitivity and specificity of the IHC test. This type of cocktail has unique technical challenges. For example, the specific antibodies in the cocktail target unique epitopes in different proteins that may have quite disparate optimal AR conditions and buffers. Combining such antibodies into one cocktail using a single AR method may result in unexpected labeling (false‐positives and false‐negatives). Thus, careful validation of this type of cocktail is important. If the diagnostic sensitivity and specificity of individual antibodies are to be compared to that of the cocktail, the same AR methods and buffers must be used for both the individual antibodies and the cocktail. Additionally, careful documentation of methods is paramount to ensure repeatability among laboratories.

Kalyuzhny has summarized the nature of a good antibody: “Having a good antibody for IHC is not only about getting a strong staining signal with low background, but also about knowing the staining makes sense in terms of its histological and physiological relevance.”28


  1. 1. Hewitt, S.M., Robinowitz, M., Bogen, S.A., et al. (2011) Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline, 2nd edn. CLSI document I/LA28‐A2. Clinical and Laboratory Standards Institute, Wayne, PA.
  2. 2. Absolom, D.R. and Van Oss, C.J. T (1986) The nature of the antigen‐antibody bond and the factors affecting its association and dissociation. Crit Rev Immunol 6:1–46.
  3. 3. Lipman, N.S., Jackson, L.R., Trudel, L.J., and Weis‐Garcia F. (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. Inst Lab Anim Res J 46:258–268.
  4. 4. Mighell, A.J., Hume, W.J., and Robinson, P.A. (1998) An overview of the complexities and subtleties of immunohistochemistry. Oral Dis 4:217–223.
  5. 5. Mandel, U., Therkildsen, M.H., Reibel, J., et al. (1992) Cancer‐associated changes in glycosylation of fibronectin: immunohistological localization of oncofetal fibronectin defined by monoclonal antibodies. APMIS 100:817–826.
  6. 6. Mandell, J.W. (2008) Immunohistochemical assessment of protein phosphorylation state: the dream and the reality. Histochem Cell Biol 130:465–471.
  7. 7. Binder, C.J., Hörkkö, S., Dewan, A., et al. (2003) Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nature Med 9:736–743.
  8. 8. Cunha‐Neto, E., Bilate, A.M., Hyland, K.V., et al. (2006) Induction of cardiac autoimmunity in Chagas heart disease: a case for molecular mimicry. Autoimmunity 39:41–54.
  9. 9. Ramos‐Vara, J.A. (2005) Technical aspects of immunohistochemistry. Vet Pathol 42:405–426.
  10. 10. Buchwallow, I.B. and Böcker W. (2010) Antibodies for immunohistochemistry. In Immunohistochemistry: Basics and Methods (eds. I. Buchwallow and W. Böcker). Springer‐Verlag, Berlin, pp. 1–8.
  11. 11. Hayat, M.A. (2002) Antigens and antibodies. In Microscopy, Immunohistochemistry, and Antigen Retrieval Methods for Light and Electron Microscopy (ed. M.A. Hayat). Kluwer Academic, Dordrecht, pp. 31–51.
  12. 12. Bogen, S.A., Vani, K., and Sompuram, S.R. (2009) Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin‐induced cross‐links. Biotech Histochem 84:207–215.
  13. 13. Sompuran, S.R., Vani, K., and Bogen, S.A. (2006) A molecular model of antigen retrieval using a peptide array. Am J Clin Pathol 125:91–98.
  14. 14. Fredenburgh, J.L. and Myers, R.B. (2002) Basic IHC workshop. 28th Annual National Society for Histotechnology Meeting, Long Beach, CA.
  15. 15. Elias, J.M. (2003) Immunohistochemical methods. In Immunohistopathology. A Practical Approach to Diagnosis, 2nd edn. (ed. J.M. Elias). ASCP Press, Chicago, IL, pp. 1–110.
  16. 16. Nelson, P.N., Reynolds, G.M., Waldron, E.E., et al. (2000) Demystified…Monoclonal antibodies. J Clin Pathol Mol Pathol 53:111–117.
  17. 17. Van Oss, C.J. and Absolom, D.R. (1984) Nature and thermodynamics of antigen‐antibody interactions. In Molecular Immunology (eds. M.Z. Atassi, C.J. van Oss, and D.R. Absolom). Marcel Dekker, New York, pp. 337–360.
  18. 18. Nelson, P.N., Fletcher, S.M., MacDonald, D., et al. (1991) Assay restriction profiles of three monoclonal antibodies recognizing the G3m(u) allotype. Development of an allotype specific assay. J Immunol Meth 138:57–64.
  19. 19. Cano, G., Milanezi, F., Leitão, D., et al. (2003) Estimation of hormone receptor status in fine‐needle aspirates and paraffin‐embedded sections from breast cancer using the novel rabbit monoclonal antibodies SP1 and SP2. Diagn Cytol 29:207–211.
  20. 20. Groves, D.J. and Morris, B.A. (2000) Veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas. Hybridoma 19:201–214.
  21. 21. Liguori, M.J., Hoff‐Velk, J.A., and Ostrow, D.H. (2001) Recombinant human interleukin‐6 enhances the immunoglobulin secretion of a rabbit–rabbit hybridoma. Hybridoma 20:189–198.
  22. 22. Rossi, S., Laurino, L., Furlanetto, A., et al. (2005) A comparative study between a novel category of immunoreagents and the corresponding mouse monoclonal antibodies. Am J Clin Pathol 124:295–302.
  23. 23. Wong, S.C.C., Chan, J.K.C., Lo, E.S.F., et al. (2007) The contribution of bifunctional SkipDewax pretreatment solution, rabbit monoclonal antibodies, and polymer detection systems in immunohistochemistry. Arch Pathol Lab Med 131;1047–1055.
  24. 24. Vilches‐Moure, J.G. and Ramos‐Vara, J.A. (2005) Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues. J Vet Diagn Invest 17:346–350.
  25. 25. Ramos‐Vara, J.A., Miller, M.A., and Valli, V.E.O. (2007) Immunohistochemical detection of multiple myeloma 1/interferon regulatory factor 4 (MUM1/IRF‐4) in canine plasmacytoma: comparison with CD79a and CD20. Vet Pathol 44:875–884.
  26. 26. Taylor, C.R. (2006) Quantifiable internal reference standards for immunohistochemistry. The measurement of quantity by weight. Appl Immunohistochem Mol Morphol 14:253–259.
  27. 27. Smedley, R.C., Lamoureux, J., Sledge, D.G., and Kiupel, M. (2011) Immunohistochemical diagnosis of canine oral amelanotic melanocytic neoplasms. Vet Pathol 48:32–40.
  28. 28. Kalyuzhny, A.E. (2009) The dark side of the immunohistochemical moon: industry. J Histochem Cytochem 57:1099–1101.

Detection methods

The antigen–antibody reaction cannot be seen with the light microscope unless it is tagged to a visible label. Therefore, labels (reporter molecules) are attached to the primary, secondary, or tertiary antibodies of a detection system to allow visualization of the immune reaction. The most commonly used labels are enzymes (e.g., peroxidase, alkaline phosphatase, glucose oxidase).1 Enzymes in the presence of a specific substrate and a chromogen produce a colored precipitate at the site of the antigen–antibody reaction.2 Most common chromogens impart a brown, red, or blue color to the reaction. The choice of enzyme and chromogen depends on several factors, such as the reaction intensity, antigen location, presence or absence of endogenous pigments, or mounting media used, but often is a matter of personal preference.3 For horseradish peroxidase, 3,3′ diaminobenzidine tetrachloride (DAB) is the most commonly used chromogen, imparting a brown color that is insoluble in organic solvents.2 For alkaline phosphatase, 5‐bromo‐4‐chloro‐3‐indolylphosphate/nitro blue tetrazoliumchloride (BCIP/NBT) (blue, permanent media), Fast red (red, aqueous mounting media), and new fuchsin (fuchsia, permanent media) are the chromogens most commonly used.2 Van der Loos has reviewed the chromogens used in multiplex IHC with photonic microscope and spectral analysis.4

The primary goal of detection in IHC is to use the fewest steps in the shortest time for visualization of the optimal immunoreactivity in tissue sections.5 In other words, the sensitivity of the detection system affects the outcome of an IHC test. In addition, the detection system must be reproducible, accurate, and render an excellent signal‐to‐noise ratio when combined with the AR method used.6 The detection system must be compatible with the animal species to be tested. Some companies marketing IHC detection systems for veterinary use optimize their detection kits based on the species evaluated (e.g., PromARKTM for dogs and cats, food animals, etc.), reducing the chances of background or false‐positive staining. The three more commonly used IHC methods are avidin–biotin based methods, non‐avidin–biotin polymer methods and catalyzed signal amplification (CSA). In these methods, the first layer of antibodies is unlabeled, but the second layer (for two‐step methods), raised against the primary antibody, is labeled. For three‐step methods, the tertiary reagent is labeled.7

Avidin–biotin methods

Avidin is a large glycoprotein extracted from egg whites that has four binding sites per molecule and high affinity for a low molecular weight vitamin called biotin. Streptavidin (from Streptomyces avidinii) produces less background than avidin. Biotin has one binding site for avidin and can be attached through other sites to an antibody (biotinylated antibody) or any other macromolecule such as an enzyme, fluorochromes or other label.7 The increased sensitivity of avidin–biotin methods results from the larger number of biotin molecules (and therefore label molecules) that can be attached to a primary antibody.8–10

One of the most common avidin–biotin methods is the avidin–biotin complex (ABC) method (Figure 3.5A). In this case, the second antibody is biotinylated and the third reagent is a complex of avidin mixed with biotin linked with appropriate label. The avidin and labeled biotin are allowed to react for about 30 minutes before application, resulting in the formation of a large complex with numerous molecules of label (e.g., enzyme). Another commonly used avidin–biotin method is the labeled avidin–biotin (LAB) or labeled streptavidin–biotin (LSAB). This method uses a biotinylated secondary antibody and a third reagent of peroxidase (or alkaline phosphatase)‐labeled avidin. The sensitivity of this method is higher than that of standard ABC.11

Schematic diagram of the avidin–biotin complex method displaying the interaction of avidin, biotin-peroxidase, biotinylated Ab, primary antibody, and antigens.
Schematic diagram of the polymer method displaying the interaction of dextran-Ig-Po complex, first Ab, and antigens.

Figure 3.5 (A) Avidin–biotin complex (ABC) method. The primary antibody (in black) binds the antigen on the tissue section followed by incubation with biotinylated antibody (in red). Avidin–peroxidase molecules then will bind biotinylated immunoglobulins. The antigen–antibody reaction is detected by a colored reaction produced when the enzyme molecules (e.g., peroxidase) interact with a substrate and a chromogen. (B) Polymer method. This detection system does not rely on the binding of avidin to biotin. The second antibody (in red) is attached to a polymer containing numerous molecules of enzyme.

(Source: Ramos‐Vara and Miller, 2014. Reproduced with permission of SAGE Publications.)

Endogenous biotin activity or tissue affinity for avidin, known as endogenous avidin–biotin activity (EABA), is common in certain tissues, particularly those rich in biotin, such as the liver, brown fat, adrenal cortex, and kidney. EABA is more prominent after HIER, although it is also present in tissues subject to other types of AR.7,12–15 EABA‐blocking reagents (pure avidin and biotin solutions) are commercially available but less expensive, “homemade” reagents are also effective (egg white solution as a source of avidin; 5% dried milk solution as a source of biotin).14,16–20

Polymeric labeling two‐step method

This is currently the most widely used non‐avidin–biotin method and the detection method of choice in many laboratories (Figure 3.5B). It consists of a compact polymer to which multiple (4–70) molecules of enzyme (peroxidase or alkaline phosphatase) and the secondary antibody specific for the primary antibody (1–10 Ig molecules) are attached.5,21 The advantages are: (1) simplicity compared to the three‐step methods; (2) equal or higher sensitivity than ABC or LSAB methods; (3) lack of background due to endogenous biotin or avidin.22–26 This method is usually more expensive than ABC or LSAB methods. Higher sensitivity can be achieved using a three‐step method, which uses a bridge antibody to link the primary antibody and the polymer complex.

Catalyzed signal amplification

This method is based on the ability of tyramide to bind to a solid substrate (e.g., tissue section) following oxidation/radicalization27 and the deposition of biotinylated or FITC‐conjugated tyramide at the location of the antigen–antibody reaction, catalyzed by horseradish peroxidase (HRP). Highly reactive intermediates formed during the HRP–tyramide reaction bind covalently to electron‐rich amino acids (e.g., tyrosine) of proteins in the vicinity of the HRP‐binding sites, via the production of free radicals by the oxygen liberated by HRP.28 This reaction is very short‐lived and, therefore, the deposition of biotinylated tyramide occurs only at or near the site where it is generated.29 The biotin conjugated to the bound tyramide is subsequently used for the attachment of avidin conjugated to HRP.29 This method is complex and laborious because it involves an initial avidin–biotin procedure followed by the tyramide reaction. However, the sensitivity can be increased 5‐ to 10‐fold compared to the regular avidin–biotin method.30 This method is suitable for antigens present in very low amounts, but background can be a problem, particularly when using HIER.31 Modifications using fluoresceinated tyramide result in marked reduction or complete disappearance of background by endogenous biotin.31–34

Immunohistochemistry on mouse tissues

With standard IHC on mouse tissues, background labeling develops due to the binding of the secondary antibody (anti‐mouse immunoglobulins) to endogenous immunoglobulins in the interstitium, plasma, B lymphocytes, and plasma cells.28,35 Commercially available mouse‐specific detection systems have eliminated this problem. Secondary antibodies to mouse IgGs may also react with Igs of other species producing the same type of nonspecific background in plasma or any tissue containing IgGs and plasma cells.


  1. 1. Ramos‐Vara, J.A. and Saeteele J. (2007) Immunohistochemistry. In Making and Using Antibodies: A Practical Handbook (eds. G.C. Howard and M.R. Kaser). CRC Press, Boca Raton, pp. 273–314.
  2. 2. Ramos‐Vara, J.A. (2005) Technical aspects of immunohistochemistry. Vet Pathol 42:405–426.
  3. 3. Van Hecke, D. (2002) Routine immunohistochemical staining today: choices to make, challenges to take. J Histotechnol 25:45–54.
  4. 4. Van der Loos, C.M. (2010) Chromogens in multiple immunohistochemical staining used for visual assessment and spectral imaging: the colorful future. J Histotechnol 33:31–40.
  5. 5. Hewitt, S.M., Robinowitz, M., Bogen, S.A., et al. (2011) Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline, 2nd edn. CLSI document I/LA28‐A2. Clinical and Laboratory Standards Institute, Wayne, PA.
  6. 6. Ramos‐Vara, J.A. and Miller, M.A. (2006) Comparison of two polymer‐based immunohistochemical detection systems: ENVISION + TM and ImmPRESSTM. J Micros 224:135–139.
  7. 7. Polak, J.M. and Van Noorden S (2003) Introduction to Immunocytochemistry, 3rd edn. Bios Scientific, Oxford.
  8. 8. Guesdon, J.L., Ternynck, T., and Avrameas, S. (1979) The uses of avidin‐biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27:1131–1139.
  9. 9. Hsu, S.‐M. and Raine, L. (1981) Protein, A., avidin and biotin in immunocytochemistry. J Histochem Cytochem 29:1349–1353.
  10. 10. Hsu, S.‐M., Raine, L., and Fanger, H. (1981) Use of avidin‐biotin‐peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580.
  11. 11. Elias, J.M., Margiotta, M., and Gaborc, D. (1989) Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin‐biotin peroxidase complex (ABC), and peroxidase‐labeled avidin‐biotin (LAB) methods. Am J Clin Pathol 92:62–67.
  12. 12. Bussolati, G., Gugliotta, P., Volante, M., et al. (1997) Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Histopathology 31:400–407.
  13. 13. Kim, D.H., Jung, K.C., Shin, Y.K., et al. (2002) The enhanced reactivity of endogenous biotin‐like molecules by antigen retrieval procedures and signal amplification with tyramine. Histochem J 34:97–103.
  14. 14. Miller, R.T. (2011) What every pathologist needs to know about technical immunohistochemistry (or how to avoid “immune confusion”). American Academy of Oral Maxillofacial Pathology Annual Meeting, San Juan, Puerto Rico, April 30.
  15. 15. Nikiel, B., Chekan, M., Jarzab, M., and Lange D. (2009) Endogenous avidin biotin (EABA) in thyroid pathology: immunohistochemical study. Thyroid Res 2:5.
  16. 16. Banerjee, D. and Pettit S. (1984) Endogenous avidin‐binding activity in human lymphoid tissue. J Clin Pathol 37:223–225.
  17. 17. Duhamel, R.C. and Johnson, D.A. (1985) Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. J Histochem Cytochem 33:711–714.
  18. 18. Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H. (1984) Improved technique utilizing nonfat dried milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal Tech 1:3–8.
  19. 19. Ramos‐Vara, J.A. (2013) Immunohistochemical methods. In Making and Using Antibodies: A Practical Handbook (eds. G.C. Howard and M.R. Kaser). CRC Press, Boca Raton, pp.303–341.
  20. 20. Wood, G.S. and Warnke, R. (1981) Suppression of endogenous avidin‐binding activity in tissues and its relevance to biotin‐avidin detection systems. J Histochem Cytochem 29:1196–1204.
  21. 21. Wiedorn, K.H., Goldmann, T., Henne, C., et al. (2001) EnVision+, a new dextran polymer‐based signal enhancement technique for in situ hybridization (ISH). J Histochem Cytochem 49:1067–1071.
  22. 22. Petrosyan, K., Tamayo, R., and Joseph, D. (2002) Sensitivity of a novel biotin‐free detection reagent (Powervision + TM) for immunohistochemistry. J Histotechnol 25:247–250.
  23. 23. Sabattini, E., Bisgaard, K., Ascani, S., et al. (1998) The EnVision TM+ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMateTM, CSA, LABC, and SABC techniques. J Clin Pathol 51:506–511.
  24. 24. Shi, S.‐R., Guo, J., Cote, R.J., et al. (1999) Sensitivity and detection efficiency of a novel two‐step detection system (PowerVision) for immunohistochemistry. Appl Immunohistochem Mol Morphol 7:201–208.
  25. 25. Taylor, C.R., Shi, S.‐R., Barr, N.J., et al. (2002) Techniques of immunohistochemistry: principles, pitfalls, and standardization. In Diagnostic Immunohistochemistry (ed. D.J. Dabbs). Churchill Livingstone, Edinburgh, pp. 3–43.
  26. 26. Vyberg, M. and Nielsen, S. (1998) Dextran polymer conjugate two‐step visualization system for immunohistochemistry. A comparison of EnVision + with two three‐step avodin‐biotin techniques. Appl Immunohistochem 6:3–10.
  27. 27. Gross, A.J. and Sizer, I.W. (1959) The oxidation of tyramine, tyrosine, and related compounds by peroxidase. J Biol Chem 234:1611–1614.
  28. 28. Buchwallow, I.B. and Böcker, W. (2010) Immunostaining enhancement. In Immunohistochemistry: Basics and Methods (eds. I. Buchwallow and W. Böcker). Springer‐Verlag, Berlin, pp. 47–59.
  29. 29. Hayat, M.A. (2002) Factors affecting antigen retrieval. In Microscopy, Immunohistochemistry, and Antigen Retrieval Methods for Light and Electron Microscopy (ed. M.A. Hayat). Kluwer Academic, Dordrecht, pp. 53–69.
  30. 30. Speel, E.J.M., Hopman, A.H.N., and Komminoth, P. (1999) Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S). J Histochem Cytochem 47:281–288.
  31. 31. Hasui, K., Takatsuka, T., Sakamoto, R., et al. (2002) Improvement of supersensitive immunohistochemistry with an autostainer: a simplified catalyzed signal amplification system. Histochem J 34:215–222.
  32. 32. Hasui, K. and Murata, F. (2005) A new simplified catalyzed signal amplification system for minimizing non‐specific staining in tissues with supersensitive immunohistochemistry. Arch Histol Cytol 68:1–17.
  33. 33. Okada, H., Iwamaru, Y., Fukuda, S., et al. (2012) Detection of disease‐associated prion protein in the optic nerve and the adrenal gland of cattle with bovine spongiform encephalopathy by using highly sensitive immunolabeling procedures. J Histochem Cytochem 60:290–300.
  34. 34. Van Gijlswijk, R.P.M., Zijlmans, H.J.M.A.A., Wiegant, J., et al. (1997) Fluorochrome‐labeled tyramines: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem 45:375–382.
  35. 35. Fung, K.‐M., Messing, A., Lee, V.M.Y., and Trojanowski, J.Q. (1992) A novel modification of the avidin‐biotin complex method for immunohistochemical studies of transgenic mice with murine monoclonal antibodies. J Histochem Cytochem 40:1319–1328.

Storage and handling of reagents

Proper handling and storage of reagents is essential for reproducibility of IHC tests. The shelf‐life of many reagents is directly linked to appropriate storage, and for many primary antibodies can be extended beyond the manufacturer’s expiration date with proper handling and storage.1–3 However, such use of a reagent must be properly documented. In general, antibodies should be stored at 0–4 °C, in the dark and in vials that do not promote immunoglobulin aggregation. For long‐term storage, freeze antibodies in aliquots at or below −20 °C in a non‐defrosting freezer.


  1. 1. Balaton, A.J., Drachenberg, C.B., Rucker, C., et al. (1999) Satisfactory performance of primary antibodies beyond manufacturer’s recommended expiration dates. Appl Immunohistochem Mol Morphol 7:221–225.
  2. 2. Savage, E.C. and DeYoung, B.R. (2010) Antibody expiration in the context of resource limitation. What is the evidence basis? Am J Clin Pathol 134:60–64.
  3. 3. Tubbs, R.R., Nagle, R., Leslie, K., et al. (1998) Extension of useful reagent shelf life beyond manufacturer’s recommendations. Arch Pathol Lab Med 122:1051–1052.

Controls in immunohistochemistry

Positive tissue control

A positive tissue control is defined as tissue known to contain the target antigen. Positive tissue controls assess the performance of the primary antibody and ideally must be fixed, processed and stained in the same way as the diagnostic case tissue for every antibody and procedure.1–3 However, time in storage and other practical factors cannot always be controlled in a diagnostic setting. Control tissues should be from the same species as the test tissue and, when possible, should be included on the same slide as the test tissue. The amount of antigen in the positive control should not be overly abundant and should have some heterogeneity in intensity through the sample; weakly immunoreactive areas can be used to detect subtle changes in antibody sensitivity.3 The presence of the antigen in the tissue control should be confirmed by another method or proven in previous publications. Internal positive tissue controls can be present in many test tissues. In the past, the detection of smooth muscle markers or vimentin in normal blood vessels suggested that fixation did not have deleterious effects on immunoreactivity for a given antigen.4,5 However, with the current antigen retrieval procedures, the statement of an “universal” marker to detect fixation problems does not hold true. We and others have demonstrated that fixation effects vary among different markers under the same fixation conditions.6 A much better assessment of the effects of fixation for a given antigen is the detection of the antigen in the test tissue (e.g., detection of CD3 in normal lymphocytes within a node with lymphoma).

Negative tissue control

A negative tissue control is tissue that is known not to contain the antigen of interest.1,3 The use of a negative control is recommended with each IHC run to verify the specificity of the primary antibody and the presence/absence of background (false‐positive) reactivity; if false‐positive reactivity (tissue labeling with a pattern similar or identical to that in the positive control) occurs in the negative control, the test should be considered invalid.1

Reagent (antibody) controls

Negative reagent controls replace the primary antibody. They are used to confirm the test specificity and to assess the degree of nonspecific background caused by the secondary (and sometimes tertiary) antibody.1 Negative reagent controls include: (1) antibody diluent; (2) same species non‐immune immunoglobulin of the same dilution and concentration; (3) an irrelevant antibody; or (4) buffer.2,3

Methods for high‐throughput evaluationof control tissues

Various methods for the simultaneous study of multiple FFPE tissues in a single histologic section have been reported. The “sausage technique” has been used heavily in the past.7 In this method, long thin strips of fixed tissues are drawn into a tube of unfixed small intestine. The entire sample is then fixed, resulting in a tight column that can be cut into blocks and embedded. Placenta as a “wrapping” tissue also has been used for this method.8 Modifications to the original method have been reported.5

Paraffin‐embedded tissue microarrays have been used as controls in human IHC laboratories, mainly for tumor diagnosis.8–12 The tissue microarray technology allows simultaneous examination of tens to hundreds of tissues on a single microscope slide. A small portion (core) of a tissue sample is taken from the “donor” paraffin block and transferred to the “recipient” block, which may contain up to 1000 cores.13 Validation studies should be carried out on multi‐tissue control blocks containing both known‐positive and known‐negative normal and neoplastic tissues.3 In veterinary medicine, species‐specific tissue microarrays are not commercially available. Species‐specific tumor cell lines are being used for IHC controls (e.g., IHC for HER‐2/neu).2,14 This approach provides an identical tissue control among different laboratories and is an excellent way to compare results.15 The advantages of the tissue microarray method include less reagent consumption, decreased technical time, decreased variability of results, the possibility of digitizing and quantifying results or interpretation of results by hierarchical cluster analysis, quality control, and standardization, evaluation of antibody sensitivity and specificity, and rapid and high‐throughput discovery and validation of biomarkers.16–20 With an adequate selection of control tissues, fewer than 12 tissue cores in an array are sufficient to evaluate more than 90% of the markers used in diagnostic IHC.20 Tissue microarray has some disadvantages when compared with the classic single‐sample microscope slide: it is technically demanding to prepare the array (with a commercial manual or automated array), and careful planning and quality control are required to ensure that cores contain representative tissue.


  1. 1. Hewitt, S.M., Robinowitz, M., Bogen, S.A., et al. (2011) Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline, 2nd edn. CLSI document I/LA28‐A2. Clinical and Laboratory Standards Institute, Wayne, PA.
  2. 2. Rasmussen, O.F. (2006) Controls. In Immunohistochemical Staining Methods, 4th edn. (ed. M. Key). Dako Corporation, Carpinteria, CA, pp. 113–118.
  3. 3. Taylor, C.R. (2006) Quantifiable internal reference standards for immunohistochemistry. The measurement of quantity by weight. Appl Immunohistochem Mol Morphol 14:253–259.
  4. 4. Miller, R.T. (2011) What every pathologist needs to know about technical immunohistochemistry (or how to avoid “immune confusion”). American Academy of Oral Maxillofacial Pathology Annual Meeting. San Juan, Puerto Rico, April 30.
  5. 5. Battifora, H. and Kopinski M. (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. J Histochem Cytochem 34:1095–1100.
  6. 6. Ramos‐Vara, J.A. and Miller, M.A. (2014) When tissue antigens and antibodies get along: Revisiting the technical aspects of immunohistochemistry, the red, brown, and blue technique. Vet Pathol 51: 42–87.
  7. 7. Fritz, K. (2010) Method for making multitissue control blocks. J Histotechnol 33(1):49–51.
  8. 8. Hsu, F.D., Nielsen, T.O., Alkushi, A., et al. (2002) Tissue microarrays are an effective quality assurance tool for diagnostic immunohistochemistry. Mod Pathol 15:1374–1380.
  9. 9. Packeisen, J., Buerger, H., Krech, R., and Boecker W. (2002) Tissue microarrays: a new approach for quality control in immunohistochemistry. J Clin Pathol 55:613–615.
  10. 10. Packeisen, J., Korsching, E., Herbst, H., et al. (2003) Demystified…Tissue microarray technology. J Clin Pathol 56:198–204.
  11. 11. Ramos‐Vara, J.A. (2005) Technical aspects of immunohistochemistry. Vet Pathol 42:405–426.
  12. 12. Simon, R. and Mirlacher, M. (2012) Tissue microarrays for translational research. In Microarrays in Diagnostics and Biomarker Development (ed. B. Jordan). Springer‐Verlag, Berlin, pp. 135–152.
  13. 13. Rhodes, A., Jasani, B., Couturier, J., et al. (2002) A formalin‐fixed, paraffin‐processed cell line standard for quality control of immunohistochemical assay of HER‐2/neu expression in breast cancer. Am J Clin Pathol 117:81–89.
  14. 14. Ramos‐Vara, J.A., Kiupel, M., Baszler, T., et al. (2008) Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories. J Vet Diagn Invest 20:393–413.
  15. 15. Jensen, T.A. and Hammond, M.E.H. (2001) The tissue microarray. A technical guide for histologists. J Histotechnol 24:283–287.
  16. 16. Nishizuka, S., Chen, S.‐T., Gwadry, F.G., et al. (2003) Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling. Cancer Res 63:5243–5250.
  17. 17. Rangel, C.S. (2002) The tissue microarray: helpful hints! J Histotechnol 25:93–100.
  18. 18. Rizzardi, A., Johnson, A.T., Vogel, R.I., et al. (2012) Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 7:42.
  19. 19. Storz, M. and Moch, H. (2013) Tissue microarrays and biomarker validation in molecular diagnostics. In Molecular Genetic Pathology (eds. L. Cheng, D.Y. Zhang, and J.N. Eble). Springer, Berlin, pp. 455–463.
  20. 20. Hewitt, S.M., Takikita, M., Abedi‐Ardekani, B., et al. (2008) Validation of proteomics based discovery with tissue microarrays. Proteom Clin Appl 2:1460–1466.

Standardization (optimization) and validation of a new IHC test

Primary antibody standardization and validation

The development of a new IHC test begins with the optimization of each of the steps in the analytical phase (phase 2) of the IHC test including: antigen retrieval, blocking nonspecific activities, and the binding and selection of primary antibody. Critical to the analytical phase is the ability of the primary antibody to specific target antigen in the host tissues for which the test is being developed.

Primary antibodies are frequently raised from human or mouse cellular targets and use in veterinary species requires caution and additional testing. Interspecies variations in antibody reactions result from subtle changes in the amino acid sequence of a given antigen and identical antibody clones that target the same antigen can differ in reactivity among species.1 An example is Melan‐A, which labels melanocytes in dogs, cats and other species, but not in horses (Figure 3.6).1 As such, additional validation of an antibody is necessary to confirm that the primary antibody is specific, selective, and reproducibly detects its target antigen.2

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Mar 30, 2020 | Posted by in INTERNAL MEDICINE | Comments Off on Immunohistochemistry

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