Improvement of supersensitive immunohistochemistry with an autostainer: a simplified catalysed signal amplification system

The ImmunoMax/catalysed signal amplification (CSA) system is a supersensitive method of paraffin immunohistochemistry. It incorporates antigen retrieval, the streptavidin–biotin complex (sABC) method, and the catalysing reporter deposition/catalysing biotinylated tyramide reaction. Strong, non-specific cytoplasmic reaction in the ImmunoMax/CSA is due to endogenous biotin unmasked in the antigen retrieval step. We examined procedures to diminish this non-specific immunoreaction and improved the ImmunoMax/CSA. Antigen retrieval in a hot water bath yielded a smaller endogenous biotin immunoreaction than antigen unmasking in an autoclave. Post-antigen retrieval fixation in buffered 10% formalin solution suppressed the biotin immunoreaction but masked the target antigen, Ki67. Post-reaction washing with 0.1% Tween 20 in Tris–HCl buffer at 35°C did not diminish the endogenous biotin immunoreaction. Animal serum also did not suppress the non-specific immunoreactivity of biotin and antibodies. Because endogenous biotin is detected by duplicated biotin–streptavidin reactions in the ImmunoMax/CSA, we replaced the sABC step with a labelled polymer secondary antibody (the EnVision system) – a simplified CSA system – because the sensitivity ofx the EnVision system was the same as that of the sABC method. The non-specific immunoreaction induced by the EnVision system was masked competitively by blocking protein. By using an antibody against Ki67 antigen that can react only with the nucleus, we were able to evaluate the non-specific cytoplasmic immunoreaction induced by the detection system. We believe that the simplified CSA system will open up the field of supersensitive paraffin immunohistochemistry.     

不同抗原修复方法对肝癌组织中内源性生物素的影响及对策

目的探讨不同抗原修复方法对肝癌组织中内源性生物素的影响以及消除办法.方法采用pH6.0、pH8.0及pH9.0不同pH值的抗原修复液进行修复,对30例经甲醛固定石蜡包埋的肝细胞癌组织中内源性生物素活性进行检测,并通过不同检测方法对30例肝细胞癌中AFP(Alpha Fetoprotein)染色结果进行对比.结果加热抗原修复暴露肝细胞癌组织中内源性生物素的活性.不同pH值抗原修复液对内源性生物素的活性影响各不相同,阳性强度随着pH值增高而增强.生物素阻断剂能有效阻断内源性生物素的活性.采用pH9.0Tris-EDTA进行修复加Envision检测系统可提高肝癌组织中AFP的阳性率及阳性强度.结论加热抗原修复可使被甲醛封闭的内源性生物素的活性重新暴露,对免疫组化的染色结果造成影响,生物素阻断系统是消除内源性生物素的有效方法,采用pH9.0的抗原修复液进行修复加Envision非生物素检测系统是免疫组化染色中较为理想的检测方法.     

Antigen retrieval,signal amplification and intensification in immunohistochemistry

In this overview we emphasize new methods of improving immunohistochemical results in formaldehyde-fixed tissue samples. The benefit of heat-induced antigen retrieval in demasking of concealed epitopes is demonstrated. We provide guidance on the influence of heat-induced antigen retrieval in commonly applied monoclonal and polyconal antibodies. Moreover, we show the promising methods of signal amplification using biotinylated tyramine and signal intensification of diaminobenzidine reaction products by metallic ions.     

Signal amplification in flow cytometry using biotin tyramine.

BACKGROUND: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents--normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to generate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. METHODS: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti-human CD4 and anti-human CD36 antibodies binding to either human lymphocytes or mixed mononuclear cells. RESULTS: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification. CONCLUSIONS: The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.     

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy.

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein-streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 microg/ml.     

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.     

Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique

Neuroendocrine cells are often disclosed in human gastric adenocarcinomas and may be recognised by their immunoreactivity towards chromogranin A. However, in dedifferentiated neuroendocrine tumour cells, the chromogranin A content may be reduced making it difficult to detect with conventional immunohistochemical methods. We therefore used a sensitive signal amplification technique in order to evaluate chromogranin A immunoreactivity and thus neuroendocrine differentiation in 40 gastric adenocarcinomas.Neuroendocrine cells were visualised by means of a monoclonal chromogranin A antibody and the avidin–biotin peroxidase complex technique, without and with addition of tyramide signal amplification. Double immunohistochemistry towards chromogranin A and Ki-67 were used to disclose proliferation in the neoplastic cells.A marked increase in the number of carcinomas containing chromogranin A-immunoreactive neoplastic cells was noted when applying the tyramide signal amplification technique. In addition, the number of immunoreactive cells within each tumour increased, and in some cases almost all the neoplastic cells became immunoreactive. Chromogranin A-immunoreactive tumour cells showing signs of proliferation were found in the majority of these carcinomas.In conclusion, we have disclosed widespread immunoreactivity towards chromogranin A in a proportion of gastric adenocarcinomas when enhancing the signal with tyramide signal amplification. Neuroendocrine differentiation is thus a common finding in gastric carcinomas when using sensitive methods.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

Multiple-reaction cycling: a method for enhancement of the immunochemical signal of monoclonal antibodies

Most current studies using immunochemical and immunohistochemical procedures to detect antigen-antibody complexes employ some type of indirect method. Such procedures afford signal amplification because several marker-conjugate molecules can bind to each primary antibody molecule. We have observed that for monoclonal antibodies an even greater amplification can be afforded simply by performing two (or more) reaction cycles (i.e., primary antibody, secondary antibody-primary antibody, secondary antibody-etc). In the present report, we demonstrate the utility of this method for immunohistochemical (immunofluorescence) and immunochemical (ELISA: enzyme-linked immunosorbent assay) procedures employing well-characterized monoclonal antibodies directed against avian type IV (basement membrane) collagen.     

Localization of human airway trypsin-like protease in the airway: an immunohistochemical study

Human airway trypsin-like protease (HAT) has been isolated from mucoid sputum of patients with chronic airway diseases. In order to clarify the cellular source of this novel protease in the human airway, we examined the localization of immunoreactive HAT in bronchial tissues obtained at surgery and fixed in 4% paraformaldehyde using an extremely sensitive immunohistochemical technique called a catalyzed signal amplification method and a monoclonal antibody against recombinant HAT. HAT immunoreactivity was demonstrated in cytoplasm of ciliated cells of bronchial epithelium and/or at the basal part of cilia. No positive reaction was found in submucosal glands or mast cells. The heterogeneous distribution of HAT immunoreactivity within the bronchial epithelium indicates that its expression might be changeable and that it might be closely related to the physiological status of the airway epithelium. Non-specific but intense reaction caused by endogenous avidin-binding activity (EABA) was selectively detected in submucosal glands, but was effectively blocked by successive treatments with avidin and biotin. These results indicate that HAT may be synthesized in the ciliated cells and that it may play some physiological roles within the epithelial layer and on the airway surface. It is necessary to keep in mind that some cells show strong EABA, especially when a highly sensitive immunohistochemical technique is applied.     

Amplification methods for the immunolocalization of rare molecules in cells and tissues

The needs to precisely assign macromolecules to specific locations and domains within tissues and cells and to reveal antigens which are present in low or even in trace amounts, led to the elaboration of a wide spectrum of immunocytochemical amplification procedures. These arise from the successive improvements of tissue preparation techniques, of antigen retrieval procedures and of immunological or non-immunological detection systems. Improvement of detection systems may be the most active in the development of amplification techniques. Since the early work of Coons, in which by the introduction of the indirect technique has started amplifying the signal, different systems have succeeded in increasing the sensitivity of antigens detection. Indeed, amplification techniques such as the multiple antibody layers, the multiple bridges, the enzyme complexes, the avidin-biotin, the silver intensification, and the numerous variations and combinations among these have increased the sensitivity for the detection of scarce tissue antigens. However, as shown by the recent progress carried out with new approaches such as the catalyzed reporter deposition (CARD) and the enhanced polymer one-step staining (EPOS), more efficient methods are still needed. In electron microscopy, few techniques have reached the resolution afforded by the post-embedding immunogold approach. In spite of this and in order to further increase its sensitivity, new probes and novel approaches are allowing combination of the gold marker with the amplification capacity of enzymes afforded by the CARD technique. Immunogold amplification strategies, such as the multiple incubations with the primary antibody and the use of an anti-protein A antibody have also led to enhanced signals displaying the advantages in terms of resolution and possibilities of quantification inherent to the colloidal gold marker.     

Microwave stimulation of an immunological reaction (CEA/anti-CEA) and its use in immunohistochemistry

Summary The effects of microwave stimulation on different aspects of immunohistochemical reactions were studied using Elisa as a model system. This technique allows monitoring of (i) the rates and recoveries of different antigen-antibody reactions, (ii) the formation of avidin/biotin complexes, (iii) the prevention of non-specific activity and (iv) the washing procedures.The binding reactions seemed to follow second-order kinetics, and reaction rates were considerably enhanced by microwave stimulation. The steps were aimed at preventing non-specific reactivity and the washing procedures could all be shortened to seconds. Such rate increases are far too large to be explained solely by the modest increase in temperature. Furthermore, microwave irradiation caused a major reduction in the yield of antigen-antibody complexes. This inhibitory effect can be compensated for by increasing the concentration of antibody. No significant effects of the microwaves on the antigen-antibody complexes were found once the complexes had been formed.The obtained results were then applied to the demonstration of carcinoembryonic antigen in histological material. By employing this method a complete immunohistochemical staining of a hydrated tissue section could be performed within 15 min, excluding the final development of colours.     

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.     

Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining.

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold-silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 microg/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold-silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.     

Quadruplex priming amplification combined with nicking enzyme for diagnostics

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.     

Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.     

Immunocytochemical characterization of mouse monoclonal ACTH antibodies with a note on staining conditions and control procedures

Summary Mouse monoclonal antibodies (MAbs) have been produced against porcine ACTH and tested for their immunocytochemical utility. Ten out of 12 MAbs reacted with formaldehyde-fixed human ACTH[1–39] and fragments thereof. Cytochemical fragment testing revealed that 6 of the 10 MAbs recognized epitopes in the vicinity of the region where porcine ACTH differs from mouse ACTH (amino acids 26, 29 and 31). Both tissue and cytochemical model data indicate that many of the MAbs detected porcine ACTH with somewhat higher potency than human and rat ACTH (rat ACTH[1–39] is identical to mouse ACTH[1–39]). MAbs Nos. 5, 8 and 12, in particular, revealed a highly satisfactory signal to noise ratio also in formalin-fixed, par-affin-embedded specimens. Most of the MAbs were potent in detecting both the high concentrations of ACTH congeners in corticotrophs and melanotrophs and the lower concentrations of such peptides in human antropyloric gastrin cells. Blocking of tissue endogenous peroxidase activity reduced reactivity towards the MAbs. This could be circumvented by use of biotinylated primary antibodies combined with avidin/streptavidin-alkaline phosphatase detection. Availability of MAbs and of the corresponding synthetic antigen also made some quantitative comparisons and analyses of appropriate control procedures possible.