Naphthol AS Phosphate Research Articles

Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes.

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes.

New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5-60 min at 37 degrees C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2-9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels.

Paired indirect immunoenzyme staining based on primary antisera from the same species was performed sequentially without intermediate antibody elution. The first antigen was labelled brown by an immunoperoxidase procedure (either the two-stage indirect method, the unlabelled antibody peroxidase-antiperoxidase method, or the avidin-biotin bridge method using diaminobenzidine (DAB) and hydrogen peroxide as the substrates. The second antigen was labelled blue by applying a two-stage indirect immuno-alkaline phosphatase procedure using naphthol AS phosphate and Fast Blue BB salt as the substrate. In this way, polyclonal mucosal immunocytes were revealed in distinctly contrasting colours when stained for kappa and lambda light chains. Glucagon and somatostatin (D) cells in human pancreatic islets, and gastrin and D cells in human gastric antral glands, were likewise clearly differentiated. Conversely, a mixed colour appeared in some immunocytes after staining for alpha and kappa chains. However, unbalanced colour mixing was sometimes difficult to interpret, and additional experiments demonstrated that unwanted interactions could take place between the two sequences of reagents if the density of the DAB deposits was insufficient. These pitfalls were incompatible with unequivocal double staining in the same cell. Nevertheless, paired staining could be conveniently applied with the described procedures when prior knowledge had established that the antigens in question were located in separate cells.

Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils.

The aim of the present paper is to detect two different antigens simultaneously in a single slide. In cryostat sections of human tonsils, B-lymphocytes of follicle mantle-bearing surface IgD were immunostained with the alkaline phosphatase method using monoclonal anti IgD. The subsequent staining for T-lymphocyte subsets (T-helper and T-suppressor lymphocytes) was performed again with the alkaline phosphatase method using one of the monoclonal antibodies OKT 4, OKT 8, Leu 3a, Leu 2a. The best results with the alkaline phosphatase method were achieved using naphthol AS phosphate and Fast Blue BB for the revelation of the first antigen and naphthol AS-BI phosphate and diazotized New Fuchsin for the second.

CYTOCHEMICAL STUDY OF ACID PHOSPHATASE IN ORAL EXFOLIATED CELLS

Cytochemical studies of acid phosphatase activity of oral mucosa cells have been carried on the frozen-dried smears employing the method of standard naphthol AS phosphate devised by Burstone. The purpose is to observe the possible interrelation between the intensity of acid phosphatase activity and some pathologic changes. The specimens were obtained from various areas of oral cavity of 71 persons. In the oral mucosa from healthy individuals, acid phosphatase activity was found to be positive in all the cells from various areas. Intense acid phosphatase reaction was seen in the cells of palate and the gingiva. Activity of the enzyme decreased in inflammatory conditions. The mechanism of enhancement of acid phosphatase activity in inflammatory condition was discussed in relation to keratinization.

Neutrophil alkaline phosphatase score in chronic granulocytic leukaemia: effects of splenectomy and antileukaemic drugs.

Staining with naphthol AS phosphate and Fast Blue BB salt has been used for the estimation of neutrophil alkaline phosphatase (NAP) scores in patients with chronic granulocytic leukaemia (CGL). The very low scores found at diagnosis rise when the disease is treated, and there is some inverse correlation between the NAP score and the absolute neutrophil count. Patients treated intensively developed high NAP scores. Elective splenectomy performed during the chronic phase of CGL is followed by a pronounced but transient neutrophilia and a concurrent striking rise in the NAP score. Similar changes were observed in patients without CGL who underwent splenectomy. These observations can be explained by assuming that newly formed neutrophils in CGL have a normal content of NAP but are rapidly sequestered in non-circulating extramedullary pools, whereas the circulating neutrophil with a typically low NAP content is a relatively aged cell which has lost enzyme activity. In subjects with or without CGL, removal of the spleen, a major site of such pooling, temporarily permits the circulation of newly formed neutrophils but eventually other organs assume the sequestering functions of the spleen. Thus the aberrations of NAP score seen in CGL might be attributable not to an intrinsic cellular defect but to an exaggeration of the granulocyte storage phenomena which also occur in subjects without CGL.

Naphthol as Phosphates as Fluorometric Substrates for Alkaline Phosphatase

Abstract The results of a study of three naphthol AS phosphates indicated that naphthol AS-BI was the best substrate for the analysis of alkaline phosphatase. With the use of naphthol AS-BL phosphate, 0.0005 to 0.5 units of alkaline phosphatase can be determined.

Histochemical study of a number of hydrolases, including a new acid phosphatase, in various tissues of men and mice.

Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO4 or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.

Alkaline phosphatase activity of incisures and nodes during degeneration and regeneration of peripheral nerve fibers

Burstone's simultaneous coupling method using Naphthol AS phosphates was employed in visualizing changes in alkaline phosphatase in the nodes and incisures of fibers during degeneration and regeneration in transected sciatic nerves of rats. In one group of transected and repaired nerves, a Millipore sheath was added, and in another the suture line was left exposed. In a third group, repair was not attempted in order to study neuroma formation and degeneration in the isolated distal stump. Degenerating fibers in distal segments of early repaired specimens and in isolated distal stumps showed a decrease in alkaline phosphatase activity, until there were only scattered clumps of stained material visible in degenerating myelin by the tenth day. Regenerating fibers showed an increase in alkaline phosphatase after 5 days in the proximal stump and the transected area. During reinnervation of the distal stump, the alkaline phosphatase staining increased as the fibers became myelinated. Increase in enzyme activity in the transected area was associated with capillary budding. Prominence of the enzyme at incisures and nodes, where neurilemma cytoplasm is relatively abundant, may have metabolic significance.

APPLICATION OF THE STEREOSPECIFIC INHIBITOR L-PHENYLALANINE TO THE ENZYMORPHOLOGY OF INTESTINAL ALKALINE PHOSPHATASE.

The intestine-specific inhibitor of alkaline phosphatase, l-phenylalanine, has been employed in the study of alkaline phosphatase in rat intestine. Five substrates were used, β-glycerophosphate, o-carboxyphenylphosphate, α-naphthyl acid phosphate, naphthol AS-BI phosphate and naphthol AS-TR phosphate. The hydrolytic rate and specific l-phenylalanine inhibition was most marked in the case of the first three substrates. When formol-Ca fixed, gum-sucrose treated sections of rat intestine were incubated in these substrates in the presence separately of 0.05 Md- and l-phenylalanine and Gomori's calcium-cobalt method was employed, a striking inhibition by the l-isomer was regularly found. Azo dye methods were applied under different conditions of time, temperature and cation concentration in some instances. The contrast was marked but not dramatic in the case of α-naphthyl acid phosphate, and it was evident but not marked in the case of naphthol AS phosphate substrates. These findings have been discussed. The alkaline phosphatase of intestinal epithelial cells (human) grown in tissue culture exhibited great sensitivity to l-phenylalanine. Rat kidney and leukocyte alkaline phosphatase were relatively insensitive under the same conditions. Morphologically, the l-phenylalanine-sensitive alkaline phosphatase in rat intestine is largely confined to the striated border of the epithelial cells. In animals maintained on a high fat diet, azo dye methods demonstrated the presence of fine granules in the region of the Golgi apparatus and terminal web in the presence of d-phenylalanine. These reactions were absent in companion sections incubated in l-phenylalanine-containing substrate solutions. The possibility of comparing the morphology of a tissue in which both test and control solutions are identical except for the configuration of one component may contribute to a more precise interpretation of the results obtained in the test section.

Alkaline Phosphatase in Leucocytes by An Azo Dye Method

Alkaline phosphatase activity was demonstrated in the cytoplasm of leucocytes of man, rat and rabbit by means of naphthol AS phosphate as the substrate and fast blue BBN as the coupling agent. The reaction was effected in Tris buffer, 0.2 M at ph 9.1, in 5-10 min at 25°C. Air-dried smears, with or without fixation in 10% neutral formalin, showed activity but those heated 1 min in water at 100°C did not.

Histochemical demonstration of acid phosphatase activity in osteoclasts.

High acid phosphatase activity was observed in osteoclasts of several species using a reproducible azo-dye technique. High activity of two distinct enzymes, acid and alkaline phosphatase, are associated with osteoclasts and osteoblasts respectivey. Althouth frozen-dried tissues are recommended for definitive studies, the enzyme techniques used give satisfactory results with cold acetone-fixed tissues. The most accurate localization of acid phosphatase in osteoclasts in controlled comparative studies is obtained with double-embedded frozen-dried undecalcified tissues in conjunction with naphthol AS-phosphates.

Histochemical Comparison of Naphthol AS-Phosphates for the Demonstration of Phosphatases

Histochemical Comparison of Naphthol AS-Phosphates for the Demonstration of Phosphatases