Antigen Retrieval Immunohistochemistry: Review and Future Prospects in Research and Diagnosis over Two Decades

As a review for the 20th anniversary of publishing the antigen retrieval (AR) technique in this journal, the authors intend briefly to summarize developments in AR-immunohistochemistry (IHC)–based research and diagnostics, with particular emphasis on current challenges and future research directions. Over the past 20 years, the efforts of many different investigators have coalesced in extending the AR approach to all areas of anatomic pathology diagnosis and research and further have led to AR-based protein extraction techniques and tissue-based proteomics. As a result, formalin-fixed paraffin-embedded (FFPE) archival tissue collections are now seen as a literal treasure of materials for clinical and translational research to an extent unimaginable just two decades ago. Further research in AR-IHC is likely to focus on tissue proteomics, developing a more efficient protocol for protein extraction from FFPE tissue based on the AR principle, and combining the proteomics approach with AR-IHC to establish a practical, sophisticated platform for identifying and using biomarkers in personalized medicine.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

Protein-embedding technique: a potential approach to standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections.

A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a 'block' that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed,paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.     

Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis.

From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. "The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue". This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of integral(Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as integral (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = integral (Pffpe)/ integral (Pf) x 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf x R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe= Pf. Further studies are designed to test the limitations of the proposed hypothesis.     

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.     

Evaluation of Antigen Retrieval Buffer Systems

The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (pH 8.0) and Tris buffer (pH 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (pH 8.0) or Tris (pH 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use.     

Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.     

Proteome analysis of microdissected formalin-fixed and paraffin-embedded tissue specimens.

Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.     

Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.     

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.     

Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST–aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.     

Androgen Receptor (AR), E-Cadherin,and Ki-67 as Emerging Targets and Novel Prognostic Markers in Triple-Negative Breast Cancer (TNBC) Patients

BackgroundTNBC is an aggressive subset of breast cancer (BC) without specific target therapy.MethodsThis observational, retrospective study included 45 cases of TNBC. The aim of this study was to evaluate the expression of the AR, E-cadherin and Ki-67 in relation to histological type, time to relapse and overall survival (OS). Immunohistochemistry (IHC) was carried out on formalin-fixed paraffin-embedded tumor samples obtained from patients defined TNBC.ResultsThe AR was positive (IHC >10%) in 26.6%. E-cadherin (CDH1) expression was considered positive if the score was ≥ 2. This expression was negative in 53.3% cases. The Ki-67 index was ≥ 20% in 37.7%. Univariate analyses showed that AR, CDH1 and Ki-67 are significantly associated with OS. Multivariate analysis showed that AR and Ki-67 expression are independent variables associated with OS. The statistical analysis showed that patients with AR negative and Ki-67 positive expression have a significant correlation with poor outcome.ConclusionsOur data suggest that the combination of AR and E-cadherin expression as well as Ki-67 status might be useful prognostic markers in TNBC. Hence, these molecular determinants could play an interesting role to classify subgroups of TNBC.     

Comparison of different antibodies for detection of progesterone receptor in breast cancer

Monoclonal antibodies directed against human estrogen receptor (ER) and progesterone receptor (PR) have been used extensively for biochemical and immunohistochemical detection of receptors independent of hormone-binding assays. These antibodies have been valuable both for experimental work and for detection of receptors in clinical breast cancer specimens. The purpose of this study was to characterize the sensitivity and specificity of different antibodies for detection of PR by immunohistochemistry (IHC) of formalin-fixed paraffin breast carcinoma sections. The panel of twelve antibodies included two new ones (PgR636 and PgR1294) produced prospectively to be resistant to formalin fixation and paraffin embedding. Fifty-nine breast carcinomas, having known PR levels by biochemical ligand-binding assay, were used to prepare multitumor paraffin-embedded tissue blocks for characterization of the PR antibodies. Of all the antibodies tested, both PgR636 and PgR1294 stained the highest percentage of breast carcinomas known to be positive by the biochemical assay (95-98%) and they exhibited the highest concordance with the biochemical assay (88-90%). The PgR636 and PgR1294 antibodies, along with one other, PR 88, also gave the highest intensity of nuclear staining, while PgR636 and PgR1294 stained the highest mean percentage of tumor cell nuclei. Antigen retrieval was not necessary for PR immunostaining by PgR636 and PgR1294 in most tumors and other tissues examined, but did slightly increase the staining intensity. The majority of the other antibodies tested were highly dependent on antigen retrieval; only PR 88 and KD 68 antibodies approached the performance of PgR636 and PgR1294 without antigen retrieval. These results indicate that PgR636 and PgR1294 are optimal antibodies for IHC detection of PR in routine paraffin tissue blocks.     

Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification.

To understand the biological relationships among various molecules, it is necessary to define the cellular expression patterns of multiple genes and gene products. Relatively simple methods for performing multi-label immunohistochemical detection are available. However, there is a paucity of techniques for dual immunohistochemical (IHC) and mRNA in situ hybridization (ISH) detection. The recent development of improved non-radioactive detection systems and simplified ISH protocols has prompted us to develop a tyramide signal amplification method for sequential multi-label fluorescent ISH and IHC detection in either frozen or paraffin-embedded tissue sections. We used this method to examine the relationship between glial cell line-derived neurotrophic factor receptor alpha2 (GFRalpha2) mRNA expression and IHC localization of its co-receptor Ret in the trigeminal ganglion of postnatal Day 0 mice. We found that approximately 70% of Ret-immunoreactive neurons possessed GFRalpha2 mRNA and virtually all GFRalpha2-expressing neurons contained Ret-immunoreactive protein. Finally, we used paraformaldehyde-fixed, paraffin-embedded sections and a monoclonal antibody against neuron-specific nuclear antigen (NeuN) to demonstrate the neuronal specificity of GFRalpha2 mRNA expression in adult mouse brain. This multi-labeling technique should be applicable to a wide variety of tissues, antibodies, and probes, providing a relatively rapid and simple means to compare mRNA and protein localization.     

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 oC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.     

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.