A tetrazolium method for non-specific alkaline phosphatase

Summary A technique for the histochemical demonstration of non-specific alkaline phosphatase was developed using a medium containing indoxyl phosphate and a tetrazolium salt, Nitro B.T. The tetrazolium salt was reduced to diformazan by the hydrogen ions released by the formation of either indigo or indigo white by reaction of the enzyme on the indoxyl phosphate.The localization in the organs investigated was similar to that obtained by the standard azo dye and lead techniques.     

Dual-enzyme cascade--an amplified method for the detection of alkaline phosphatase

A method in which a two-enzyme cascade is used for rapid and sensitive detection of alkaline phosphatase is described. A masked inhibitor, 4-(3-oxo-4,4,4-trifluorobutyl)phenyl phosphate, is dephosphorylated by the action of alkaline phosphatase. The resulting compound, 1,1,1-trifluoro-4-(4-hydroxyphenyl)-butan-2-one, acts as a potent inhibitor of the second enzyme, a liver carboxylesterase. A determination of the residual esterase activity provides a highly sensitive indication of the original phosphatase concentration. The sensitivity of this dual-enzyme cascade is approximately 125-fold greater than that observed for the direct detection of phosphatase activity with p-nitrophenyl phosphate.     

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.     

Alkaline phosphatase in human lymphocytes. II. A method for ultracytochemical detection of alkaline phosphatase in lymphocytes

For the ultracytochemical identification of alkaline phosphatase in lymphocytes gained from the peripheral blood of healthy individuals a sensitive method is described which allows the low enzyme activity of these cells to be determined. This was possible because the authors succeeded in stabilizing lead ions in the alkaline medium by forming a complex directly between tris-(hydroxymethyl) aminomethan and lead (II) citrate. AP localized ultrachemically in lymphocytes in particular formations similar to phosphasomes of neutrophilic granulocytes. In those lymphocytes stimulated by lipopolysaccharides a high enzyme activity could be observed and, in addition to phosphasomes, the product of response can also be found in canal-like structures of the endoplasmatic reticulum. These findings contribute to clarify the ultrastructural localization of alkaline phosphatase in lymphocytes and may be regarded as an aid in discovering the importance of the enzyme in the biology of lymphocytes or in its activation, respectively.     

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

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.     

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.     

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)     

Chemiluminescent detection of alkaline phosphatase in PhastGel.

A chemiluminescent assay has been applied to the detection of alkaline phosphatase on PhastGel containing lysates of preimplantation mouse embryos. The very sensitive detection capabilities reported for the chemiluminescent system led to the investigation of its applicability to the characterization of the alkaline phosphatases in one embryo or less and to compare the sensitivity of two different commercial alkaline phosphatase chemiluminescent assays to a colorimetric assay.     

Multiple epitope labeling by the exclusive use of alkaline phosphatase conjugates in immunohistochemistry

 Here we demonstrate a simple and reliable multiple epitope labeling technique based exclusively on the alkaline phosphatase (AP) enzyme-linked visualization method. AP is functionally blocked by ethylenediaminetetraacetic acid (EDTA), which allows the repeated use of AP conjugates in immunohistochemistry with different substrates. We found that reactivation of AP function following EDTA incubation is dependent on EDTA concentration and incubation time. While incubation times of up to 2 h in 0.25 M EDTA, pH 6, exhibit a resumption of AP enzyme function, more than 2 h of incubation irreversibly blocks AP enzyme activity. Surprisingly, EDTA incubation also results in considerable but not complete inhibition of antibody crossreactivity during immunohistochemistry. Thus, this technique is suitable for single-layer, multiple-staining experiments with AP-linked primary antibodies or multilayer labeling with antibodies of various species for sequential staining rounds. We demonstrate the applicability of this technique in immunohistochemistry by double-labeling experiments using the monoclonal antibodies anti-glial fibrillary acidic protein, anti-leucocyte common antigen, anti-CD43/CD45RA (pan-human leucocyte), and anti-migration inhibitory factor-related protein-8 in combination with an in situ nick translation assay to characterize differentiating antigens of apoptotic cells in human glioblastoma paraffin sections. Accepted: 2 April 1998     

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 new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.     

An improved fluorogenic substrate for the detection of alkaline phosphatase activity

We designed a new alkaline phosphatase (ALP)-sensitive fluorogenic probe in which a self-immolative spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic compound to improve substrate specificity. Enzymatic hydrolysis converts the fluorogenic substrate 1 to a highly fluorescent reporter 3, thus allowing for the fast and quantitative analysis of ALP activity with greatly increased affinity for the enzyme.     

Salinity-sensitive alkaline phosphatase activity in gills of the blue crab,Callinectes sapidus Rathbun

A levamisole-sensitive (K_i = 0.72 mM) alkaline phosphatase (pH optimum 9.1) and a levamisole-insensitive alkaline phosphatase (pH optimum 7.1) are present in gills of the blue crab Callinectes sapidus. Both enzymes are distinct from ouabain-sensitive ATPase. Specific activity for either phosphatase is greatest in the acinar tissue, which lines the branchial vessels. Histochemical localization of the enzymes confirmed this distribution. Activity of levamisole-sensitive alkaline phosphatase is affected by acclimation salinity. V_max of the levamisole-sensitive alkaline phosphatase is greater in high-salinity crabs than in low-salinity crabs; apparent K_m is not significantly different. The levamisole-sensitive alkaline phosphatase associated with the acinar tissue lining the branchial vessels may modulate the osmoregulatory response in blue crabs.     

An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.     

A method for determining alkaline phosphatase activity in marine phytoplankton using spectrofluorometry

A method for determining relative percent intensity alkaline phosphatase activity (APA) using enzyme labeled fluorescence coupled with spectrofluorometry is presented. Compared to traditional microscopy and flow cytometry, we increase statistical power and reduce sample-handling issues. Combined with a biological standard, our method can quantify APA of natural plankton assemblages.