Recent advances in catalytic peroxidase histochemistry.

Immunoassays have developed to become an important analytical tool in life sciences for detection of endogenous and exogenous targets. Among the most important enzyme labels horseradish peroxidase (HRP), alkaline phosphatase (AP), and beta-D-galactosidase (GAL) is HRP the smallest enzyme and plays nowadays an outstanding role. The oldest substrates are chromogens widely applied for localization of sites of peroxidase (PO) activity in histochemistry as well as for colorimetric applications. They are represented by a diversity of aromatic amines and phenols. Encouraged by development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels represent the most sensitive and worthwhile detection tools to date. In contrast to chromogens fluorescent labels for detection PO activity are confined only to a few substrates developed more recently. These substrates are mostly applied in histochemistry at a short time scale due to their frequently high solubility. At the long time scale sole exception is so far the tyramine based fluorochome deposition technique (more general: catalytic reporter deposition, CARD). Despite quite different staining behavior both fluorometric and product deposition related principles are based on 4-hydroxy phenylalkyl substrates. The following article reviews basic principles of peroxidatic substrate degradation processes including chromogenic and fluorescent approaches with emphasis on recent advances in development of chromogens and fluorogens for application in histology. As a result of systematic efforts towards the design of substrates, the range of classical precipitating chromogens as well as fluorescent techniques could be complimented by novel highly sensitive substrates with superior staining capabilities: a) Metal chelating 2-hydroxy benzylamines are derived from classical aniline substrates (two steps) and utilize metal catalytic effects in an efficient intramolecular way. The enzymatically yielded dark colored polycondensation products are applicable in histochemistry, in colorimetry and especially as precipitating electron opaque labels with enhanced osmiophilic properties for light and electron microscopy. b) Fluorescent 4-hydroxy-styryl derivatives are capable of oxidative selfanchoring reactions at the cellular level close to sites of PO activity. In contrast to deposition of tyramine conjugated fluorochromes an altered fluorochrome with improved fluorescence properties is furnished during oxidative crosslinking of the substrate. This results in a highly specific and photostable fluorescence response and an outstanding low background staining. Histochemical and immunohistochemical applications are presented.     

Histochemical studies of acid and alkaline phosphatases in rat tooth germs with undecalcified resin-embedded specimens.

A novel technique for the histochemical demonstration of acid phosphatase (AcPase) and alkaline phosphatase (AkPase) in hard tissues has been proposed. Fresh, unfixed, undecalcified samples of rat tooth germs and surrounding structures were embedded in LR Gold resin at -20 degrees C. Sections of 2 microns were taken and subsequently processed for enzyme histochemistry. AkPase reaction product appeared as strong linear staining outlining cell boundaries and was present in the enamel organ, dental pulp, and osteoblast cells. Tartrate-resistant AcPase staining was seen exclusively in the osteoclasts of developing alveolar bone. Our results demonstrated that the use of unfixed, undecalcified LR Gold resin-embedded specimens for histochemistry is a novel technique which may be of value for certain studies when decalcification of specimens is undesirable. The technique appears to give good preservation of enzyme activity combined with the ability to prepare sections with excellent morphological detail.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.     

Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as “reactive-tracers” for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.     

Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as “reactive-tracers” for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.     

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)     

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 image analysis for enzyme histochemical and immunocytochemical staining

Immunocytochemical and enzyme histochemical analyses of cells and tissues are used to detect changes in the extent of injury and the expression of various molecules. Image analysis quantitation offers an easier, more efficient technique to evaluate these changes. We studied the application of image analysis for evaluating enzyme histochemistry and immunocytochemistry of cells and tissues as a way to assess stroke. Using brain sections, we compared investigator and computer-generated image analysis of 2,3,5-triphenyltetrazolium chloride stained cerebral infarcts in rats subjected to 2 h middle cerebral artery occlusion and 22 h re-perfusion. Both methods documented the infarct volumes with a comparison of means of less then 5%. This suggests no difference between computer- and hand-calculated values. Computer-generated analysis was easier and faster to use. Using endothelial cell monolayers, immunocytochemical staining of a time course of heat shock protein expression was compared to a grading system using fast red chromagen counterstained with hematoxylin. Results demonstrated greater ease and efficiency with computer-generated image analysis compared to other subjective systems of analysis. Image analysis is more useful for detecting small differences in staining, especially when using 3,3-diaminobenzidine as a chromagen. Investigator bias is also reduced using this system. Our comparisons validate the use of this versatile technology to assess more easily both cell and tissues in stroke research.     

Validation of image analysis for enzyme histochemical and immunocytochemical staining

Immunocytochemical and enzyme histochemical analyses of cells and tissues are used to detect changes in the extent of injury and the expression of various molecules. Image analysis quantitation offers an easier, more efficient technique to evaluate these changes. We studied the application of image analysis for evaluating enzyme histochemistry and immunocytochemistry of cells and tissues as a way to assess stroke. Using brain sections, we compared investigator and computer-generated image analysis of 2,3,5-triphenyltetrazolium chloride stained cerebral infarcts in rats subjected to 2 h middle cerebral artery occlusion and 22 h re-perfusion. Both methods documented the infarct volumes with a comparison of means of less then 5%. This suggests no difference between computer- and hand-calculated values. Computer-generated analysis was easier and faster to use. Using endothelial cell monolayers, immunocytochemical staining of a time course of heat shock protein expression was compared to a grading system using fast red chromagen counterstained with hematoxylin. Results demonstrated greater ease and efficiency with computer-generated image analysis compared to other subjective systems of analysis. Image analysis is more useful for detecting small differences in staining, especially when using 3,3-diaminobenzidine as a chromagen. Investigator bias is also reduced using this system. Our comparisons validate the use of this versatile technology to assess more easily both cell and tissues in stroke research.     

Cepaea hortensis agglutinin-I, specific for O-glycosidically linked sialic acids, selectively labels endothelial cells of distinct vascular beds

The lectin Cepaea hortensis agglutinin-I (CHA-I) binds to O-glycosidically linked sialic acids with previously characterized specificity. Employing histochemistry, we demonstrate that CHA-I is a useful probe for detecting sialic acids in formalin-fixed human tissues in a specific manner. It stains the endothelium of arteries and veins in all tissues examined, and labels the capillaries in distinct vascular beds including the brain, colon, thyroid, pituitary, and adrenal. By contrast, the endothelial sinusoids in the liver, spleen, and bone marrow remained unstained. The staining pattern of CHA-I overlaps with the distribution of the sialomucin and L-selectin ligand podocalyxin, which includes positivity of podocytes and interstitial but not glomerular capillaries. CHA-I-positive epithelial structures were found in the lung, liver and kidney. Colon carcinoma cells were labelled with CHA-I but not haemangiosarcomas. In summary, CHA-I is a useful tool for detecting O-glycosidically linked sialic acids in formalin-fixed tissues, and a potentially powerful tool for the isolation and characterization of unknown sialomucins in normal and eventually in diseased tissues.     

New technique for processing paraffin-embedded tissue for Y chromosome fluorescence identification of trophoblastic disease

A new reprocessing technique for Y chromosome fluorescent body (q 12 region) detection of trophoblastic disease in previously paraffin-embedded tissues is described. Deparaffinized sections were treated with pronase and trypsin for digestion, followed by hydrolysis with HCl and acetic acid, staining with quinacrine hydrochloride fluorochrome and mounting in S?rensen's phosphate buffer (pH 5.5). Use of the technique resulted in sufficient fluorescence quality and better accuracy for Y and X heterochromatin scoring. The technique yielded the same results in retrospective formalin-fixed, paraffin-embedded trophoblastic specimens as in fresh tissues. The combinations of enzymes and acids and the dosages necessary for optimal results are discussed.     

Advantages of histochemistry for the study of cell biology

Summary Bridging between the two bodies of cell science, histochemistry has brought together knowledge of morphological structures and biochemical constituents and provided insight into the function of one or the other. Where it has localized an enzyme, hormone or other entity of known biological activity to a cell type, histochemistry has contributed insight into the cell's function. By detecting heterogeneity in the content of an enzyme or glycoconjugate within a presumed uniform population of cells, the histochemical approach has differentiated among these cells subtypes with demonstrated or presumed differences in function. On the other hand, in instances where it has located an isozyme, antigen, glycoconjugate or other entity of uncertain significance in a cell or organelle of known function, histochemistry has suggested a possible role for the constituent related to that of the structure. Histochemical examination provides the observer with a different view of body tissues and their composition from that obtained by strictly morphological or chemical techniques. Information acquired through this advantage is cited wherein an immunohistochemical method disclosed previously undiscovered neural organs and carbohydrate histochemistry detected previously unrecognized glycoconjugates. Presented as the David Glick Lecture at the 9th International Congress of Histochemistry and Cytochemistry in Maastricht The Netherlands, 30 August–5 September, 1992.     

Immunocytochemical localization of a tartrate-resistant and vanadate-sensitive acid nucleotide tri- and diphosphatase

Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel-free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate-sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis.     

人骨形成蛋白1-cDNA基因工程表达产物抗体的制备及鉴定

应用在大肠杆菌中表达的人骨形成蛋白(human bone morphogenetie protein,HBMP-1)的N端片段作为免疫原,免疫新西兰大白兔,成功地制备了抗人骨形成蛋白的抗血清,经免疫转移电泳证实该抗血清有较好的特异性,用ABC免疫组织化学方法在正常和恶性肿瘤骨的石腊切片上检测抗血清效价为1:100～1:1000,免疫组化染色结果表明,HBMP存在于人胎下颌骨新生的骨质和骨细胞中,颌骨骨肉瘤和软骨肉瘤的瘤细胞呈BMP强阳性反应,这一结果表明,骨的生长、形成和骨肿瘤的发生与BMP密切相关。     

Use of laser microprobe mass analysis (LAMMA) for localizing multiple elements in soft and hard tissues

The potential of laser microprobe mass analysis (LAMMA) as a sensitive microanalytical technique was explored in applications relevant to nephrology. Aluminum and associated elements, such as iron, were localized in fresh tissue biopsies obtained from uremic patients treatment by chronic hemodialysis. The LAMMA was applied to serum, liver, bone, and parathyroid glands of such patients. In addition, we used LAMMA to evaluate the specificity and sensitivity of routine histochemistry, in particular on human bone sections stained by the aluminon method. The high, multielemental sensitivity and molecular microprobe potential of LAMMA established important advantages over other microchemical methods forin situ analysis at the micron level in histological sections.     

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.     

Detection of myelination using a novel histological probe.

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease.     

Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy

Various forms of green fluorescent protein (GFP) have become important reporters of gene transfer and expression after transfection or infection of cells in cell culture. Frequently, molecular biological assays (Northern blots, PCR) are applied to detect reporter gene expression in target organs. However, these methods are not suitable for evaluation of tissue- or cell-specific expression which would be of great interest especially in case of using tissue-specific promoters. Therefore, organs of transgenic mice with the enhanced green fluorescent protein (EGFP) gene under control of the cytomegalovirus (CMV) promoter were processed for histology by formaldehyde fixation and embedding in paraffin. Sections were deparaffinized, mounted and evaluated for fluorescence in a confocal laser scanning microscope. This method combines the advantages of direct exploitation of tissue sections without further staining procedures with evaluable tissue-, cell-, and even subcellular-specific distribution patterns of EGFP expression in tissues. Results obtained by direct evaluation of EGFP fluorescence in paraffin sections were confirmed by immunohistochemical staining with anti-EGFP. In the present report, we demonstrate that application of confocal microscopy on routinely processed histological preparations is very suitable for determining gene transfer efficiency and promotor activities.     

A new preparation method for several histopathological examinations on a single block.

A new method of specimen preparation is described permitting several studies such as routine staining, histochemistry, enzyme histochemistry, immunohistochemistry, and electron microscopy on a single block of biopsy specimens. Tissues are immersed in the fixative, which primarily stabilizes carbohydrate moieties, and embedded in the mixture of JB-4, methylmethacrylate and divinylbenzene. The resin is polymerized at 4 C. Thin sections (1-2 microns) are obtained with a sliding microtome, and ultrathin sections (60-90 millimicrons) with a ultramicrotome. The sections are stained directly with various conventional procedures without removing the embedding resin. This preparation method offers a potentially useful tool for histopathological studies on biopsy specimens.