Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.     

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Defining the molecular target of an antibody derived from nuclear extract of Jurkat cells using protein arrays

Many diagnostic antibodies are generated by immunization with whole cells or cell extracts and are shown by screening on tissue sections to label specific cell populations. However, their target molecule then needs to be identified, and this can be technically demanding. Here we describe the use of protein arrays to define the targets of new or uncharacterized monoclonal antibodies. The technique involves screening protein arrays containing thousands of recombinant human proteins. An initial experiment showed that a well-characterized monoclonal antibody against nucleophosmin identified 22 clones on the array encoding this protein. Next, the antibody JJ166, for which the antigen had not yet been identified, was screened. This antibody was generated by immunizing with a nuclear extract of Jurkat cells and was detected in immunohistochemistry due to its distinctive nuclear staining of lymphoid tissue cells. However, its molecular target had remained unidentified using traditional approaches. A protein array screen rapidly identified the mitotic spindle-associated molecule NUMA1 (nuclear mitotic apparatus protein 1). To confirm this putative specificity, JJ166 was shown to react with COS-1 cells transiently transfected with the complementary DNA for NUMA1. Furthermore, a commercially available antibody against NUMA1 showed nearly identical staining in immunohistochemistry on human tissue and cells. Overall, this method represents an effective and quick strategy for defining the protein targets of new or uncharacterized monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.     

Monoclonal antibody CD133-2 (AC141) against hematopoietic stem cell antigen CD133 shows crossreactivity with cytokeratin 18.

CD133 is an antigen expressed on hematopoietic progenitor cells and on some epithelial cells. We previously reported that a commercially available antibody against CD133, CD133-2/AC141, also reacted with an intracellular protein in placental trophoblasts. Here we show by 2D electrophoresis and mass spectroscopy that this reactivity is with cytokeratin 18, a cytokeratin present in most simple epithelia. Immunohistochemistry (IHC) with CD133-2/AC141 on a trophoblast cell line displayed a staining pattern typical for the cytoskeleton. Cryostat sections of stratified epithelia lacking cytokeratin 18 did not react with CD133-2/AC141. In conclusion, care must be taken not to misinterpret staining patterns using CD133-2/AC141 in IHC.     

IHC Profiler: An Open Source Plugin for the Quantitative Evaluation and Automated Scoring of Immunohistochemistry Images of Human Tissue Samples

In anatomic pathology, immunohistochemistry (IHC) serves as a diagnostic and prognostic method for identification of disease markers in tissue samples that directly influences classification and grading the disease, influencing patient management. However, till today over most of the world, pathological analysis of tissue samples remained a time-consuming and subjective procedure, wherein the intensity of antibody staining is manually judged and thus scoring decision is directly influenced by visual bias. This instigated us to design a simple method of automated digital IHC image analysis algorithm for an unbiased, quantitative assessment of antibody staining intensity in tissue sections. As a first step, we adopted the spectral deconvolution method of DAB/hematoxylin color spectra by using optimized optical density vectors of the color deconvolution plugin for proper separation of the DAB color spectra. Then the DAB stained image is displayed in a new window wherein it undergoes pixel-by-pixel analysis, and displays the full profile along with its scoring decision. Based on the mathematical formula conceptualized, the algorithm is thoroughly tested by analyzing scores assigned to thousands (n = 1703) of DAB stained IHC images including sample images taken from human protein atlas web resource. The IHC Profiler plugin developed is compatible with the open resource digital image analysis software, ImageJ, which creates a pixel-by-pixel analysis profile of a digital IHC image and further assigns a score in a four tier system. A comparison study between manual pathological analysis and IHC Profiler resolved in a match of 88.6% (P<0.0001, CI = 95%). This new tool developed for clinical histopathological sample analysis can be adopted globally for scoring most protein targets where the marker protein expression is of cytoplasmic and/or nuclear type. We foresee that this method will minimize the problem of inter-observer variations across labs and further help in worldwide patient stratification potentially benefitting various multinational clinical trial initiatives.     

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.     

Mechanisms of immunity to rickettsial infection: characterization of a cytotoxic effector cell

Rickettsiae, as other intracellular bacteria, are relatively sequestered from the effects of antibody and local antibody-independent responses. Considering the obligate intracellular nature of rickettsia, the exact mechanisms by which lymphocytes and macrophages encounter rickettsial antigens and eliminate the infection depends upon the appropriate presentation of antigen to the immune system. We demonstrate here that cells taken from the spleens of Rickettsia typhi- or R. tsutsugamushi-infected mice are able to lyse specifically tissue culture targets infected with the homologous organism. This effect was eliminated upon treatment of the spleen cells with anti-Thy-1.2 + complement. Furthermore such T cells exhibit H-2-restricted killing when tested on infected targets of different genetic backgrounds. We propose that a T cell-mediated cytotoxic immune mechanism exists that may play an important role in the elimination of rickettsial organisms during infection.     

Characterization of monoclonal antibody reacting exclusively against intracellular Orientia tsutsugamushi

Intracellular bacteria often change the expression of their genes in order to adapt to new environmental conditions. Here we describe a monoclonal antibody (MAb) that reacts exclusively against intracellular Orientia tsutsugamushi. Although MAb applied to the 56-kDa protein, a major outer membrane protein, reacted against a large number of bacteria that had attached to host cells at the early stage of infection, M686-13 reacted against only a minor portion of the attached bacteria. In the later stage of the intracellular growth cycle, both antibodies showed identical staining patterns by double immunofluorescent staining. These results suggest that M686-13 reacted to an epitope or a protein that had probably been expressed during the intracellular growth cycle and rapidly diluted or degraded upon release into the extracellular environment. Although its molecular characteristics remain unknown, the reactive antigen may prove to be a novel developmental antigen and this MAb could be used as reagent for the staining of viable O. tsutsugamushi.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

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.     

Widespread occurrence of avian spectrin in nonerythroid cells

We have prepared an antibody against chicken erythrocyte α spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with α spectrin in chicken erythrocytes and crossreacts with α spectrin in erythrocytes from various mammals. Immunofluorescence reveals that this antiserum reacts with a plasma membrane component in erythrocytes as well as in most nonerythroid avian and mammalian cells. Intense staining is seen at or near the plasma membrane in neurons, lens cells, endothelial and epithelial cells of the gastrointestinal and respiratory tracts, skeletal and cardiac muscle, as well as skeletal myotubes grown in tissue culture. Immunoautoradiography indicates that the crossreactive antigen in these nonerythroid tissues has the same molecular weight and isoelectric point as the chicken erythrocyte antigen. Smooth muscle, tracheal cilia, myelin and mature sperm stain weakly or not at all. These results suggest that spectrin is more extensively distributed than previously recognized, and that the functions of spectrin elucidated for erythrocytes may apply to other cell types as well.     

Lack of Specificity of Commercially Available Antisera: Better Specifications Needed

The ideal antiserum for immunohistochemical (IHC) applications contains monospecific high-affinity antibodies with little nonspecific adherence to sections. Many commercially available antibodies are “affinity” purified, but it is unknown if they meet “hard” specificity criteria, such as absence of staining in tissues genetically deficient for the antigen or a staining pattern that is identical to that of an antibody raised against a different epitope on the same protein. Reviewers, therefore, often require additional characterization. Although the affinity-purified antibodies used in our study on the distribution of muscarinic receptors produced selective staining patterns on sections, few passed the preabsorption test, and none produced bands of the anticipated size on Western blots. More importantly, none showed a difference in staining pattern on sections or Western blots between wild-type and knockout mice. Because these antibodies were used in most studies published thus far, our findings cast doubts on the validity of the extant body of morphological knowledge of the whole family of muscarinic receptors. We formulate requirements that antibody-specification data sheets should meet and propose that journals for which IHC is a core technique facilitate consumer rating of antibodies. “Certified” antibodies could avoid fruitless and costly validation assays and should become the standard of commercial suppliers. (J Histochem Cytochem 56:1099–1111, 2008)     

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.     

Bodian's silver impregnation of endocrine cells

Summary We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Bodian's silver impregnation of endocrine cells. A tentative explanation to the staining mechanism

We have recently shown that a variety of proteins, including albumin and immunoglobulins conjugated to colloidal gold, strongly binds to certain basic peptide sequences, and neurohormonal peptides. Silver proteinate, used in the classical Bodian's neurohistological procedure, is now shown to bind to the same peptide sequences in cytochemical model systems. In tissue, gastrin cells and glucagon cells have been reported to show strong unspecific immunocytochemical staining and these cell types also stain in the Bodian's procedure. These results suggest that certain types of unspecific immunocytochemical staining and the Bodian's silver staining method may depend upon a common mechanism, involving binding of labelled or aggregated protein to basic and hydrophobic sequences in tissue.     

Identification of a 70 kD protein with sequence homology to squid neurofilament protein in glial cells of the leech CNS

A monoclonal antibody G39, generated against a protein extract of leech central nervous system, labels specific cell types in adult, embryonic, and regenerating preparations. The antibody stained glial cells, microglial cells, and connective tissue cells, but nor neurons or muscle on cryosections. The staining pattern resembled that of an intracellular network. Affinity purification of the antigen revealed a 70 kD protein. Peptide sequencing showed significant homology of a stretch of 15 amino acids to squid neural filament protein. The same mAb G39 delineated glial cells as they formed during development of the CNS and showed that the giant neuropil glial cells appear before those in the packets. The antigen recognized by mAb G39 represents a nonneuronal intermediate filament of the leech Hirudo medicinalis found in various cell-types such as glia, microglia, and some cells of the connective tissue. 1994 John Wiley & Sons, Inc.