Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

Summary We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique

We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.     

Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging.

Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse, and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

Improvements in histological quality and signal retention following in situ hybridization in early chick embryos using plastic resin and recolorization.

We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.     

An immunogold-silver staining method for detection of cell surface antigens in cell smears

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.     

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.     

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

Menadiol diphosphate,a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry

Summary Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce³⁺ ions and the DAB−Ni−H₂O₂ procedure yielded better results than Ca²⁺ ions in the Co−(NH₄)₂S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce³⁺ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT. Supported by the Alexander von Humboldt-Stiftung and the Deutsche Forschungsgemeinschaft (Sfb 174)     

A new colorimetric method for determination of creatine phosphokinase

A new colorimetric method has been developed for the determination of creatine phosphokinase (CPK). This method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with p-nitrophenylglyoxal (PNPG) under alkaline conditions to produce a colored product which absorbs maximally at 480 nm. At 25 degrees C the reaction was complete after 10 min in 0.1 M sodium pyrophosphate, pH 12, containing 0.15 M sodium ascorbate. The colored product was stable for at least 24 h and obeyed Beer's law in the range 0.005-0.05 mM creatine. The color reaction was used to determine the activity of CPK in serum and tissue extracts. The results obtained by this method agreed well with the results obtained by other available methods for CPK determination. However, the PNPG method was more rapid and more sensitive than other colorimetric methods and required a single chromogenic reagent.     

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)     

骨髓标本中免疫组织化学及DNA原位末端标记双重染色技术的应用

为扩展免疫酶标记分子病理学技术在血液病基础与临床研究中的应用,我们探索了碱性磷酸酶抗碱性磷酸酶（A-PAAP）免疫组化和DNA原位末端标记（ISEL）技术在骨髓涂片和切片（乙二醇甲基丙烯酸酯GMA冷包埋）中联合应用（双重染色）的方法,结果令人满意,已初步用于白血病分化抗原及细胞凋亡的共同检测。     

大鼠肺泡Ⅱ型上皮细胞的原代分离

目的建立大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial cells typeⅡ,AECⅡ)分离、纯化、原代培养及鉴定的方法。方法用4.2U/ml的弹性蛋白酶通过气管插管注入肺泡内,消化分离AECⅡ。把细胞悬液接种到包被有大鼠IgG的塑料平皿中纯化细胞。用电镜、碱性磷酸酶显色法、改良巴氏染色法、单宁酸染色法、免疫组化染色法鉴定AECⅡ。结果细胞纯度达到90%以上,倒置显微镜下可见细胞呈岛屿状生长。电镜下可见细胞内有大量板层小体,包膜上有绒毛结构。碱性磷酸酶染色法(BCIP/NBT)可见胞浆内有蓝色颗粒。改良巴氏染色法、单宁酸染色法可见胞浆内有黑色颗粒。抗大鼠肺泡表面活性蛋白A(surfactant protein A,SP-A)免疫组化染色呈阳性反应。结论弹性蛋白酶作用温和,不损伤胞膜,分离所得细胞活力好;IgG免疫粘附纯化法操作简单,纯化效率高。电镜、BCIP/NBT、巴氏染色、单宁酸染色及免疫组化染色等鉴定方法稳定可靠,特异性高。     

A model for formulation of protein assay.

The principle of the protein assay using the reaction of an alkaline copper-protein complex with the Folin-Ciocalteu phenol reagent has been investigated. In contrast to the long-established Lowry method, a stable and rapid protein assay is developed without a buffering agent in alkaline copper solution. In the absence of a buffering agent, the reaction pH drops relatively rapidly and moves the reaction toward a more stable pH. When the reaction of alkaline copper-protein complex with Folin-Ciocalteu reagent is started at around pH 11.7, the reaction color absorbance reaches a plateau in approximately 10 min and remains stable to allow a reliable measurement of the absorbance. In the absence of the buffering agent sodium carbonate, the alkaline copper solution is also stable for months. The principle of the protein assay is presented as a model that can be used to formulate protein assays of desired specification.     

Tetrazolium methods for the histochemical investigation of hydrolases (author's transl)]

Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranito BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues. By means of NBT or TNBT as a tetrazolium salt acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase-beta-D-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme. Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction. -DS- and TC-NBT are inferior to NBT, TNBT or BSPT.     

An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Kinetic determination of alkaline phosphatase activity based on hydrolytic cleavage of the P-F bond in monofluorophosphate and fluoride ion-selective electrode

Alkaline phosphatase catalyzes the hydrolytic cleavage of the P-F bond in monofluorophosphate with the subsequent release of fluoride ions. A kinetic potentiometric method is described in which a fluoride ion-selective electrode is used for the sensitive and selective measurement of the released F- for the determination of alkaline phosphatase activity. It is shown that monofluorophosphate can be used as an alternative substrate for alkaline phosphatase. The reaction demonstrates a well-defined correlation with the hydrolysis of the P-O bond in 4-nitrophenyl phosphate. The serum alkaline phosphatase was determined in human serum samples by the potentiometric technique, and the results obtained compared well with a standard spectrophotometric method.     

Development of a novel DNA chip based on a bipolar semiconductor microchip system

We have applied an integrated circuit photodiode array (PDA) chip system to a DNA chip. The PDA chip system, constructed using conventional bipolar semiconductor technology, acts as a solid transducer surface as well as a two-dimensional photodetector. DNA hybridization was performed directly on the PDA chip. The target DNA, the Bacillus subtilis sspE gene, was amplified by polymerase chain reaction (PCR). The 340-bp PCR product was labeled using digoxigenin (DIG). A silicon nitride layer on the photodiode was treated with poly-L-lysine to immobilize the DNA on the surface of the photodiode detection elements. Consequently, the surface of the photodiode detector became positively charged. An anti-DIG-alkaline phosphatase conjugate was reacted with the hybridized DIG-labeled DNA. A color reaction was performed based on the enzymatic reaction between nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) staining solution and a DNA complex containing antibodies. A blue precipitate was formed on the surfaces of the photodiode detection elements. Successful quantitative analysis of the hybridized PCR products was achieved from the light absorption properties of the blue enzymatic reaction product that was produced after a series of reaction processes. Our DNA chip system avoids the complicated optical alignments and light-collecting optical components that are usually required for an optical DNA chip device. As a result, a simple, compact, portable and low-cost DNA chip is achieved. This system has great potential as an alternative system to the conventional DNA reader.